Compositions and methods for treating hiv/aids with immunotherapy

ABSTRACT

Chimeric antigen receptors (CARs) containing HIV envelope antigen binding domains are disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen binding fragments, and pharmaceutical compositions, relating to the CARs are also disclosed. Methods of treating or preventing HIV-infection in a subject, and methods of making CAR T cells are also disclosed. Results of treating or preventing HIV-infection, and results of making CAR T cells are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. Section119(e) to U.S. Provisional Patent Application No. 62/608,479, filed onDec. 20, 2017, U.S. Provisional Patent Application No. 62/660,819, filedon Apr. 20, 2018, the entire contents of each of which are incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Dec. 18, 2018, isnamed SequenceListing.txt and is 232 kilobytes in size.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable

FIELD OF THE DISCLOSURE

This application relates to the field of disease associated withinfection with the human immunodeficiency virus/acquiredimmunodeficiency syndrome (HIV, HIV/AIDS), particularly to proteinbinding domains and chimeric antigen receptors (CARs) containing suchbinding domains and methods of use thereof.

BACKGROUND

Infection with Human Immunodeficiency Virus (HIV) remains a major threatto human health. In 2016, 1.0 million people died from HIV-relatedcauses globally, and an estimated 36.7 million people are living withHIV. Fifty-four percent of adults and 43% of children living with HIVare currently receiving lifelong anti-retroviral drug-based therapy(ART). There is no cure for HIV infection, however ART has allowed manyto live long and productive lives (all data from WHO Fact sheet, updatedJuly 2017). Long-term anti-retroviral therapy (ART) can lead toincreased chances of severe toxicities and co-morbidities such as,lipodystrophy, insulin resistance, cardiovascular disease, and organfailure. In addition, long-term ART can lead to emergence of HIVresistance and reduced drug efficacy. Although ART is successful incontrolling infection, it is not a cure.

HIV infection can be blocked by inhibiting its ability to enter andreplicate in the host. As an alternative to ART, several HIV therapieshave been attempted that are based on passive immunotherapy using HIV-1specific broadly neutralizing antibodies to inhibit and control HIVinfection (reviewed in Margolis et al., Immunol Rev 2017; 275: 313-323).These therapies have exceptional breadth and potency against diverse HIVclades in vitro. However, these monotherapies when applied in vivo arenot fully effective and are met with several challenges such as antibodystability (reviewed in Boesch et al., Current Opinion in HIV and AIDS2015, 10(3):160-169), lack of virologic control (Bar et al., N Engl JMed 2016; 375:2037-2050), HIV resistance (Wu et al., Journal of Virology2012; 86:5844-5853), and persistence of the latent HIV reservoir(reviewed in Margolis et al., Immunol Rev 2017; 275:313-323).

Chimeric Antigen Receptors (CARs) are hybrid molecules comprising threeessential units: (1) an extracellular antigen-binding motif, (2)linking/transmembrane motifs, and (3) intracellular T-cell signalingmotifs (Long A H, Haso W M, Orentas R J. Oncoimmunology 2013; 2(4):e23621). The antigen-binding motif of a CAR is commonly fashionedafter a single chain Fragment variable (ScFv), the minimal bindingdomain of an immunoglobulin (Ig) molecule. Alternate antigen-bindingmotifs, such as receptor ligands (i.e., IL-13 has been engineered tobind tumor expressed IL-13 receptor), intact immune receptors,library-derived peptides, and innate immune system effector molecules(such as NKG2D or CD4) also have been engineered into CARs. Alternatecell targets for CAR expression (such as NK or gamma-delta T cells) arealso under development (Brown C E et al., Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012; 7 (2):e31210). Thereremains significant work to be done with regard to defining the mostactive T-cell population to transduce with CAR vectors, determining theoptimal culture and expansion techniques, and defining the moleculardetails of the CAR protein structure itself.

The linking motifs of a CAR can be a relatively stable structuraldomain, such as the constant domain of IgG, or designed to be anextended flexible linker. Structural motifs, such as those derived fromIgG constant domains, can be used to extend the ScFv binding domain awayfrom the T-cell plasma membrane surface. This may be important for somecellular targets where the binding domain is particularly close to thetumor cell surface membrane (such as for the disialoganglioside GD2;Orentas et al., unpublished observations). To date, the signaling motifsused in CARs always include the CD3-ζ chain because this core motif isthe key signal for T cell activation. The first reportedsecond-generation CARs featured CD28 signaling domains and the CD28transmembrane sequence. This motif was also used in third-generationCARs containing CD137 (4-1BB) signaling motifs (Zhao Y et al J Immunol.2009; 183 (9): 5563-74). With the advent of new technology, theactivation of T cells with beads linked to anti-CD3 and anti-CD28antibodies, and the presence of the canonical “signal 2” from CD28 wasno longer required to be encoded by the CAR itself. Using beadactivation, third-generation vectors were found to be not superior tosecond-generation vectors in in vitro assays, and they provided no clearbenefit over second-generation vectors in mouse models of leukemia (HasoW, Lee D W, Shah N N, Stetler-Stevenson M, Yuan C M, Pastan I H,Dimitrov D S, Morgan R A, FitzGerald D J, Barrett D M, Wayne A S,Mackall C L, Orentas R J. Anti-CD22-CARs targeting B cell precursor ALL,Blood. 2013; 121 (7):1165-74; Kochenderfer J N et al., Blood 2012; 119(12):2709-20). In addition to CD137, other tumor necrosis factorreceptor superfamily members such as OX40 also are able to provideimportant persistence signals in CAR-transduced T cells (Yvon E et al.Clin Cancer Res. 2009; 15(18):5852-60). Equally important are theculture conditions under which the CAR T-cell populations were cultured,for example the inclusion of the cytokines IL-2, IL-7, and/or IL-15(Kaiser A D et al. Cancer Gene Ther. 2015; 22(2):72-78).

T-cell-based immunotherapy featuring CARs has become a new frontier insynthetic biology in both cancer therapy and in therapy for infectiousdiseases. Multiple promoters and gene products can be introduced intoprimary lymphocytes using gene engineering techniques such as the use oflentivirus-based gene vectors (LV). Engineering T cells with LVexpressing CARs are envisioned to steer these highly potent cells tosites of HIV-infection and HIV-reactivation from latency, whereCAR-engineered T cells can both evade HIV infection by inhibiting fusionwith HIV virions or infected cells and also mediate effective killing ofcells expressing the viral envelope protein and thus eliminatingHIV-infected cells and thus the viral reservoir.

The “Berlin patient” (an HIV-infected bone marrow transplant patientthat received hematopoietic stem cells (HSC) from a CCR5 variant donor)is the only reported case of an HIV cure, and in principle demonstratesthat HIV eradication is achievable. One major barrier in the search foran HIV cure is the ability of HIV to persist in a latent state withincellular reservoirs in the host. One approach called “shock and kill”exploits latency reversing agents (LRAs) to reactivate the latent HIVreservoir. These agents “shock” the latently infected cell to reactivatethe virus at the epigenetic level. When combined with immunotherapiesthat effectively “kill” HIV-infected cells, this combinatorial approachhas been proposed as a pathway towards a HIV cure.

Previously tested immunotherapies using CAR strategies for HIV includefusing the CD4-receptor or a broadly-neutralizing antibody domain (bnAb)to transmembrane and the intracellular domain of the CD3zeta chain. Inthe late 1990s, first-generation anti-HIV CARs were constructed with theextracellular region of the CD4 receptor (CD4-zeta) and demonstrated tobe highly effective in eliminating HIV-infected cells in vitro (Tran etal., The Journal of Immunology 1995; 155:(2)1000-1009; Yang et al., PNAS1997; 94(21):11478-11483). However, in vivo they showed no significantcontrol over HIV infection and were possibly susceptible to HIVinfection themselves (reviewed in Lam et al. Immunotherapy 2013,5(4):404-414). Similarly, CARs engineered with bnAbs have also shownpromising results in vitro. In 2015, one group reported that an anti-HIVCAR consisting of the CD4 receptor fused to a bnAb (17b) effectivelykills HIV-infected cells while resisting infection (Liu et al., J.Virol. 2015; 89:13 10 6685-6694). One major drawback with developingCARs with bnAbs is that they require further engineering to account forreduced therapeutic effectiveness (Bar et al., N Engl J Med 2016;375:2037-2050; Sievers et al., Current Opinion in HIV and AIDS; 2015:10(3), 151-159). Furthermore, this approach may lead to undesired viralescape (Wu et al., Journal of Virology 2012; 86:5844-5853; Barr et al. NEngl J Med 2016; 375:2037-2050; Lynch et al., 2015 Journal of Virology;89(8): 4201-4213).

With regard to clinical experience with CAR-transduced T cells for HIV,the first clinical trials almost two decades ago were done using CAR-Tfor HIV. The clinical trials demonstrated that anti-HIV CAR-T therapiesare safe and have exceptional in vivo persistence (Mitsuyasu et al.,Blood 2000; 96(3): 785-793; Deeks et al., Mol Ther. 2002; 5:788). Onestriking finding was that despite only modest control of HIV viremia,modified-CAR T-cells trafficked to tissues that harbor the latent HIVreservoir (Mitsuyasu et al., Blood 2000; 96(3): 785-793). This is amajor advantage over non-CAR approaches and suggests that CAR-T activelyengages in immunosurveillance activities. Since then, studies suggestthat CAR-T may also be able to reactivate the latent HIV reservoir ofchronically-infected cells through cytokine release (Sahu et al.,Virology 2013; 446(0): 268-275). These findings have strong implicationsfor a HIV cure and support the rationale for developing improvedanti-HIV CAR-T therapies.

New HIV therapies combining HIV-1 entry or fusion inhibitors to form aChimeric Antigen Receptor (Anti-HIV CAR) is another attractive approachto effectively target and subsequently kill HIV-infected cells. Theadvantage of this approach is that in the absence of ART, anti-HIV CARshave the potential to mediate immunosurveillance of the latent HIVreservoir while effectively eliminating HIV infected cells. Furthermore,combining multiple entry and/or fusion inhibitors makes it harder forHIV to infect the CAR-modified T cell and to thereby undergo furtherreplication and develop resistance. Therefore, newly developed anti-HIVCAR therapies especially those containing more than one inhibitor havestrong implications toward a functional cure.

There is an urgent and long felt need in the art for discovering novelcompositions and methods for treatment of HIV/AIDS using an approachthat can exhibit specific and efficacious anti-HIV disease effectwithout the aforementioned short comings.

The present invention addresses these needs by providing CARcompositions and therapeutic methods that can be used to treat HIV andother diseases and/or conditions. In particular, the present inventionas disclosed and described herein provides CARs that may be used for thetreatment of diseases, disorders or conditions associated withexpression of HIV envelope protein in which CARs contain multiple HIVantigen binding domains that exhibit a high surface expression ontransduced T cells, exhibit a high degree of cytolysis of HIV-infectedcells, and in which the transduced T cells demonstrate in vivo expansionand persistence.

SUMMARY

Novel anti-HIV envelope protein antibodies or antigen binding domainsthereof and chimeric antigen receptors (CARs) that contain such anti-HIVenvelope protein antigen binding domains are provided herein, as well ashost cells (e.g., T cells) expressing the receptors, and nucleic acidmolecules encoding the receptors. The CARs exhibit a high level ofsurface expression on transduced T cells, exert a high degree ofcytolysis, and with the ability of transduced T cells to expand andpersist in vivo. Methods of using the disclosed CARs, host cells, andnucleic acid molecules are also provided, for example, to treat HIVinfection or AIDS, or HIV-related cancers in a patient.

In one aspect, improved second generation CARs are provided hereincomprising three unique classes of HIV peptide inhibitors (mD1.22,m36.4, and C46 peptide). When these domains are engineered in certainorientations within the context of an HIV Chimeric Antigen Receptor,they form a novel collection of highly potent bispecific (a combinationof two HIV inhibitors) and trispecific (a combination of three HIVinhibitors) anti-HIV CARs. The anti-HIV CARs provided herein aredesigned to destroy HIV-infected cells while conferring protection tothe CAR T-cell.

Thus, in one aspect, an isolated polynucleotide encoding an anti-HIVenvelope protein (anti-HIV binder, or simply anti-HIV) or a fragmentthereof is provided comprising a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1, 3, and 5.

In another aspect, an isolated polynucleotide encoding an anti-HIVenvelope protein linked to a second anti-HIV envelope protein, or afragment thereof is provided comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 45, 49, 53, 57, 61, 65, 75, 79,83, and 87.

In another aspect, an isolated polynucleotide encoding an anti-HIVenvelope protein is expressed in a cell encoding a second anti-HIVenvelope protein, or fragment thereof is provided comprising a nucleicacid sequence consisting of SEQ ID NO: 69.

In yet another aspect, an isolated polynucleotide encoding an anti-HIVenvelope protein linked to two additional anti-HIV envelope proteins, ora fragment thereof is provided comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 91, 95, and 99.

In another aspect, an isolated polynucleotide encoding an anti-HIVenvelope protein linked to another anti-HIV envelope protein expressedin a cell where another such anti-HIV envelope protein is expressedcomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 103, 111, 115, and 119.

In one embodiment, an isolated polynucleotide encoding a fully humananti-HIV antibody or a fragment thereof is provided, wherein theantibody or a fragment thereof comprises a fragment selected from thegroup consisting of a Fab fragment, a F(ab′)₂ fragment, a Fv fragment,and a single chain Fv (ScFv).

In one embodiment, an isolated polynucleotide encoding an anti-HIVantibody or a fragment thereof or another anti-HIV binding protein isprovided, wherein the antibody or a fragment thereof, comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 4,and 6.

In another embodiment, an isolated polynucleotide encoding an anti-HIVantibody or a fragment thereof or other anti-HIV binding protein isprovided, wherein the antibody or a fragment thereof, or other anti-HIVbinding protein is linked to a second such anti-HIV binder comprised ofan amino acid sequence selected from the group consisting of SEQ ID NOs:46, 50, 54, 58, 62, 66, 76, 80, 84, and 88.

In yet another embodiment, an isolated polynucleotide encoding ananti-HIV antibody or a fragment thereof or other anti-HIV bindingprotein is provided, wherein the antibody or a fragment thereof, orother anti-HIV binding protein is expressed in a cell that contains asecond anti-HIV binder comprised of an amino acid sequence comprisingSEQ ID NO: 70. In yet another embodiment, an isolated polynucleotideencoding an anti-HIV antibody or a fragment thereof or other anti-HIVbinding protein is provided, wherein the antibody or a fragment thereof,or other anti-HIV binding protein is linked to two additional suchanti-HIV binders comprised of an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 92, 96, and 100.

In another embodiment, an isolated polynucleotide encoding an anti-HIVenvelope protein linked to another anti-HIV envelope protein expressedin a cell where another such anti-HIV envelope proteins is expressedcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 104, 112, 116, and 120.

In one aspect, an isolated nucleic acid molecule encoding a CAR isprovided comprising, from N-terminus to C-terminus, at least one HIVantigen binding domain encoded by a nucleotide sequence comprising anucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, and 5; at least one transmembrane domain; and at least oneintracellular signaling domain.

In one embodiment, an isolated nucleic acid molecule encoding the CAR isprovided wherein the encoded extracellular HIV antigen binding domaincomprises at least one single chain variable fragment of an antibody ora minimized single antibody domain (for example VH-only) that binds tothe HIV envelope protein.

In another embodiment, an isolated nucleic acid molecule encoding theCAR is provided wherein the encoded extracellular HIV antigen bindingdomain comprises at least one heavy chain variable region of an antibodythat binds to HIV.

In yet another embodiment, an isolated nucleic acid molecule encodingthe CAR is provided wherein the encoded CAR extracellular HIV antigenbinding domain further comprises at least one lipocalin-based antigenbinding antigen (anticalins) that binds to HIV.

In yet another embodiment, an isolated nucleic acid molecule encoding aCAR is provided wherein the encoded CAR extracellular HIV antigenbinding domain is comprised of a single immunoglobulin domain, such as aVH-only domain, or a similar single chain Ig-like binding moiety.

In one embodiment, an isolated nucleic acid molecule is provided whereinthe encoded extracellular HIV antigen binding domain is connected to thetransmembrane domain by a linker domain.

In another embodiment, an isolated nucleic acid molecule encoding theCAR is provided wherein the encoded HIV extracellular antigen bindingdomain is preceded by a sequence encoding a leader or signal peptide.

In yet another embodiment, an isolated nucleic acid molecule encodingthe CAR is provided comprising at least one HIV antigen binding domainencoded by a nucleotide sequence comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1, 3, and 5, andwherein the CAR additionally encodes an extracellular antigen bindingdomain which targets an antigen that includes, but is not limited to, aHIV latency-associated antigen (e.g., CD32a), Hepatitis B Virus (HBV)surface antigen (HBsAg), Hepatitis C Virus (HCV) E2 protein,Cytomegalovirus (CMV) glycoprotein B, CD20, CD22, ROR1, mesothelin,CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4,c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR, MAGEA3 TCR, or any combination thereof.

In certain embodiments, an isolated nucleic acid molecule encoding theCAR is provided wherein the additionally encoded extracellular antigenbinding domain comprises an anti-CD32a ScFv antigen binding domain,anti-HBsAg ScFv antigen binding domain, an anti-HCV E2 ScFv antigenbinding domain, an anti-CMV glycoprotein B ScFv antigen binding domain,an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigenbinding domain, an anti-ROR1 ScFv antigen binding domain, ananti-mesothelin ScFv antigen binding domain, an anti-CD33 ScFv antigenbinding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123(IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen bindingdomain, an anti-BCMA (CD269) ScFv antigen binding domain, an anti-GPC2ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain,an anti-FGFR4 ScFv antigen binding domain, an anti-TSLPR ScFv antigenbinding domain an anti-c-Met ScFv antigen binding domain, an anti-PMSAScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen bindingdomain, an anti-EGFRvIII ScFv antigen binding domain, an anti-GD-2 ScFvantigen binding domain, an anti-NY-ESO-1 TCR ScFv antigen bindingdomain, an anti-MAGE A3 TCR ScFv antigen binding domain, or an aminoacid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof,or any combination thereof.

In one aspect, the CARs provided herein further comprise a single linkeror spacer domain.

In another aspect, the CARs provided herein that contain more than oneHIV antigen binder, may contain two, three or four linker or spacerdomains.

In one embodiment, an isolated nucleic acid molecule encoding the CAR isprovided wherein the extracellular HIV antigen binding domain, theintracellular signaling domain, or both are connected to thetransmembrane domain by a linker or spacer domain.

In one aspect, an isolated nucleic acid molecule encoding the CAR isprovided wherein the extracellular HIV antigen binding domains areconnected to one another or the transmembrane domain by nucleic acidsequence encoding a linker or spacer domain selected from the groupconsisting of SEQ ID NOs: 9, 23, 25, 27, 29, and 31.

In one embodiment, an isolated nucleic acid molecule encoding the CAR isprovided wherein the extracellular HIV antigen binding domains areconnected to one another or the transmembrane domain by a linker orspacer domain by amino acid sequence selected from the group consistingof SEQ ID NOs: 10, 24, 26, 28, 30, and 32.

In one aspect, an isolated nucleic acid molecule encoding the CAR isprovided wherein the extracellular HIV antigen binding domains areconnected to one another or the transmembrane domain by nucleic acidsequence encoding a linker or spacer domain that contains furin cleavagesites flanking a translational skip site as exemplified in SEQ ID NOs:33.

In one embodiment, an isolated nucleic acid molecule encoding the CAR isprovided wherein the extracellular HIV antigen binding domains areconnected to one another or the transmembrane domain by a linker orspacer domain that contains furin cleavage sites flanking atranslational skip site comprised of amino acid sequence as exemplifiedby SEQ ID NOs: 34.

In one embodiment, an isolated nucleic acid molecule encoding the CAR isprovided wherein the encoded linker domain is derived from theextracellular domain of CD8 or CD28, and is linked to a transmembranedomain.

In another embodiment, an isolated nucleic acid molecule encoding theCAR is provided wherein the encoded CAR further comprises atransmembrane domain that comprises a transmembrane domain of a proteinselected from the group consisting of the alpha, beta or zeta chain ofthe T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF19,or a combination thereof.

In yet another embodiment, an isolated nucleic acid molecule encodingthe CAR is provided wherein the encoded intracellular signaling domainfurther comprises a CD3 zeta intracellular domain.

In another embodiment, an isolated nucleic acid molecule encoding theCAR is provided wherein the encoded at least one intracellular signalingdomain comprises a costimulatory domain, a primary signaling domain, ora combination thereof.

In yet another embodiment, where more than one HIV antigen binders maybe expressed on two different transmembrane proteins in the same cell,the two proteins may express identical intracellular signaling domains,different signaling domains, or one may express no signaling domains.

In further embodiments, an isolated nucleic acid molecule encoding theCAR is provided wherein the encoded at least one costimulatory domaincomprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5,ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB(CD137), or a combination thereof.

In one embodiment, an isolated nucleic acid molecule encoding the CAR isprovided that further contains a leader sequence or signal peptidewherein the leader or signal peptide nucleotide sequence comprises thenucleotide sequence of SEQ ID NO: 35 or SEQ ID NO: 37.

In yet another embodiment, an isolated nucleic acid molecule encodingthe CAR is provided wherein the encoded leader sequence comprises theamino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 38.

In one aspect, a CAR is provided herein comprising, from N-terminus toC-terminus, at least one HIV antigen binding domain, at least onetransmembrane domain, and at least one intracellular signaling domain.

In one embodiment, a CAR is provided wherein the extracellular HIVantigen binding domain comprises at least one single chain variablefragment of an antibody that binds to the antigen, or at least one heavychain variable region of an antibody that binds to the antigen, or acombination thereof.

In another embodiment, a CAR is provided wherein the at least onetransmembrane domain comprises a transmembrane domain of a proteinselected from the group consisting of the alpha, beta or zeta chain ofthe T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, TNFRSF19, or acombination thereof.

In some embodiments, the CAR is provided wherein CAR additionallyencodes an extracellular antigen binding domain comprising a HIVlatency-associated antigen (e.g., CD32a), Hepatitis B Virus (HBV)surface antigen (HBsAg), Hepatitis C Virus (HCV) E2 protein,Cytomegalovirus (CMV) glycoprotein B, CD19, CD20, ROR1, mesothelin,CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4,TSLPR, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, TSLPR, NY-ESO-1 TCR,MAGE A3 TCR, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%or 99% identity thereof, or any combination thereof.

In one embodiment, the CAR is provided wherein the extracellular antigenbinding domain comprises an anti-CD32a ScFv antigen binding domain,anti-HBsAg ScFv antigen binding domain, an anti-HCV E2 ScFv antigenbinding domain, an anti-CMV glycoprotein B ScFv antigen binding domain,an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigenbinding domain, an anti-ROR1 ScFv antigen binding domain, ananti-mesothelin ScFv antigen binding domain, an anti-CD33 ScFv antigenbinding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123(IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen bindingdomain, an anti-BCMA (CD269) ScFv antigen binding domain, an anti-GPC2ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain,an anti-FGFR4 ScFv antigen binding domain, anti-TSLPR ScFv antigenbinding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSAScFv antigen binding domain, an anti-glycolipid F77 ScFv antigen bindingdomain, an anti-EGFRvIII ScFv antigen binding domain, an anti-GD-2 ScFvantigen binding domain, an anti-NY-ESO-1 TCR ScFv antigen bindingdomain, an anti-MAGE A3 TCR ScFv antigen binding domain, or an aminoacid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof,or any combination thereof.

In another embodiment, a CAR is provided wherein the at least oneintracellular signaling domain comprises a costimulatory domain and aprimary signaling domain.

In yet another embodiment, a CAR is provided wherein the at least oneintracellular signaling domain comprises a costimulatory domaincomprising a functional signaling domain of a protein selected from thegroup consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1(CD11a/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or acombination thereof.

In one embodiment, the nucleic acid sequence encoding a CAR comprisesthe nucleic acid sequence of SEQ ID NO: 39 (LTG1944,LP-mD1.22-CD8TM-41BB-CD3zeta nucleic acid sequence (FIG. 2A)). In oneembodiment, the nucleic acid sequence encodes a CAR comprising the aminoacid sequence of SEQ ID NO: 40 (LTG1944, LP-mD1.22-CD8TM-41BB-CD3zetaCAR amino acid sequence (FIG. 2A)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 41 (LTG1945,LP-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG. 2B)). In oneembodiment, the nucleic acid sequence encodes a CAR comprising the aminoacid sequence of SEQ ID NO: 42 (LTG1945, LP-m36.4-CD8TM-41BB-CD3zeta CARamino acid sequence (FIG. 2B)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 43 (LTG2328,LP-C46-CD8TM-41BB-CD3zeta CAR nucleotide sequence (FIG. 2C)). In oneembodiment, the nucleic acid sequence encodes a CAR comprising the aminoacid sequence of SEQ ID NO: 44 (LTG2328, LP-C46-CD8TM-41BB-CD3zeta CARamino acid sequence (FIG. 2C)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 47 (LTG2325,LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG.2D)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 48 (LTG2325,LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG.2D)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 51 (LTG2313,LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG.2E)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 52 (LTG2313,LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG.2E)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 55 (LTG1946,LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG.2F)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 56 (LTG1946,LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG.2F)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 59 (LTG2326,LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG.2G)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 60 (LTG2326,LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG.2G)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 63 (LTG1947,LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG.2H)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 64 (LTG1947,LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG.2H)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 67 (LTG1948,LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG.2I)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 68 (LTG1948,LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG. 2I).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 71 (LTG2303,LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CAR nucleicacid sequence (FIG. 2J)). In one embodiment, the nucleic acid sequenceencodes a CAR comprising the amino acid sequence of SEQ ID NO: 72(LTG2303, LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2CAR amino acid sequence (FIG. 2J)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 73 (LTG2322,LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM CAR nucleic acidsequence (FIG. 2K)). In one embodiment, the nucleic acid sequenceencodes a CAR comprising the amino acid sequence of SEQ ID NO: 74(LTG2322, LP-mD1.22-CD8TM-41BB-CD3 zeta-F2AF-m36.4-TNFRSF19TM CAR aminoacid sequence (FIG. 2K)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 77 (LTG2314,LP-mD1.22-L3-C46-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG.2L)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 78 (LTG2314,LP-mD1.22-L3-C46-CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG. 2L)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 81 (LTG2315,LP-mD1.22-L5-C46-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG.2M)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 82 (LTG2315,LP-mD1.22-L5-C46-CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG. 2M)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 85 (LTG2316,LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG.2N)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 86 (LTG2316,LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG. 2N)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 89 (LTG2317,LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta CAR nucleic acid sequence (FIG.2O)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 90 (LTG2317,LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta CAR amino acid sequence (FIG. 2O)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 93 (LTG2318,LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3zeta CAR nucleic acid sequence(FIG. 2P)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 94 (LTG2318,LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3zeta CAR amino acid sequence(FIG. 2P)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 97 (LTG2319,LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence(FIG. 2Q)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 98 (LTG2319,LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence(FIG. 2Q)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 101 (LTG2320,LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta CAR nucleic acid sequence(FIG. 2R)). In one embodiment, the nucleic acid sequence encodes a CARcomprising the amino acid sequence of SEQ ID NO: 102 (LTG2320,LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta CAR amino acid sequence(FIG. 2R)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 105 (LTG2323,LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM CAR nucleicacid sequence (FIG. 2S)). In one embodiment, the nucleic acid sequenceencodes a CAR comprising the amino acid sequence of SEQ ID NO: 106(LTG2323, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM CARamino acid sequence (FIG. 2S)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 107 (LTG2329,LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CARnucleic acid sequence (FIG. 2T)). In one embodiment, the nucleic acidsequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:108 (LTG2329,LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CARamino acid sequence (FIG. 2T)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 109 (LTG2330,LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CARnucleic acid sequence (FIG. 2U)). In one embodiment, the nucleic acidsequence encodes a CAR comprising the amino acid sequence of SEQ ID NO:110 (LTG2330,LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2 CARamino acid sequence (FIG. 2U)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 113 (LTG2331,LP-C46-L3-mD1.22-CD8TM-CD3zeta-F2AF-m36.4-TNFRSF19TM CAR nucleic acidsequence (FIG. 2V)). In one embodiment, the nucleic acid sequenceencodes a CAR comprising the amino acid sequence of SEQ ID NO: 114(LTG2331, LP-C46-L3-mD1.22-CD8TM-CD3zeta-F2AF-m36.4-TNFRSF19TM CAR aminoacid sequence (FIG. 2V)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 117 (LTG2332,LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM CAR nucleicacid sequence (FIG. 2W)). In one embodiment, the nucleic acid sequenceencodes a CAR comprising the amino acid sequence of SEQ ID NO: 118(LTG2332, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM CARamino acid sequence (FIG. 2W)).

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 121 (LTG2334,LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM CAR nucleicacid sequence (FIG. 2X)). In one embodiment, the nucleic acid sequenceencodes a CAR comprising the amino acid sequence of SEQ ID NO: 122(LTG2334, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM CARamino acid sequence (FIG. 2X)).

In one aspect, the CARs disclosed herein are modified to express orcontain a detectable marker for use in diagnosis, monitoring, and/orpredicting the treatment outcome such as for monitoring the progress ofsuch treatment.

In one embodiment, the nucleic acid molecule encoding the disclosed CARscan be contained in a vector, such as a viral vector. The vector is aDNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpesvirus vector, a measles virus vector, a lentiviral vector, adenoviralvector, or a retrovirus vector, or a combination thereof.

In certain embodiments, the vector further comprises a promoter whereinthe promoter is an inducible promoter, a tissue specific promoter, aconstitutive promoter, a suicide promoter, a synthetic promoter, or anycombination thereof.

In yet another embodiment, the vector expressing the CAR can be furthermodified to include one or more operative elements to control theexpression or function of CAR T cells (e.g., induciblehomo-/hetero-dimerized CAR), or to eliminate CAR-T cells by virtue of asuicide switch. The suicide switch can include, for example, anapoptosis inducing signaling cascade or a drug that induces cell death.In a preferred embodiment, the vector expressing the CAR can be furthermodified to express an enzyme such thymidine kinase (TK) or cytosinedeaminase (CD). In another aspect, host cells including the nucleic acidmolecule encoding the CAR are also provided. In some embodiments, thehost cell is a T cell, such as a primary T cell obtained from a subject.In one embodiment, the host cell is a CD8⁺ T cell.

In yet another aspect, a pharmaceutical composition is providedcomprising an anti-HIV effective amount of a population of human Tcells, wherein the T cells comprise a nucleic acid sequence that encodesa CAR, wherein the CAR comprises at least one extracellular antigenbinding domain comprising an anti-HIV antigen binding domain orcombination of domains comprising the amino acid sequence of SEQ ID NO.2, 4, 6, or combination of domains comprising the amino acid sequence ofSEQ ID NO. 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104,112, 116, and 120, at least one linker domain, at least onetransmembrane domain, and at least one intracellular signaling domain,wherein the T cells are T cells of a human having an HIV infection, or Tcells intended to be administered to a patient having HIV/AIDS.

In one embodiment, a pharmaceutical composition is provided wherein theat least one transmembrane domain of the CAR contains a transmembranedomain of a protein selected from the group consisting of the alpha,beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4,CD5, CD8, CD9, CD16, CD22, Mesothelin, CD33, CD37, CD64, CD80, CD83,CD86, CD134, CD137, CD154, TNFRSF19, or a combination thereof.

In another embodiment, a pharmaceutical composition is provided whereinthe human disease includes infection with HIV/AIDS or HIV-associatedcancers including an adult carcinoma comprising oral and pharynx cancer(tongue, mouth, pharynx, head and neck), carcinomas associated withinfection with Kaposi's sarcoma virus (HHV8), digestive system cancers(esophagus, stomach, small intestine, colon, rectum, anus, liver,interhepatic bile duct, gallbladder, pancreas), respiratory systemcancers (larynx, lung and bronchus), bones and joint cancers, softtissue cancers, skin cancers (melanoma, basal and squamous cellcarcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma,osteosarcoma, Ewing's sarcoma), tumors of the central nervous system(brain, astrocytoma, glioblastoma, glioma), and cancers of the breast,the genital system (uterine cervix, uterine corpus, ovary, vulva,vagina, prostate, testis, penis, endometrium), the urinary system(urinary bladder, kidney and renal pelvis, ureter), the eye and orbit,the endocrine system (thyroid), and the brain and other nervous system,or any combination thereof.

In yet another embodiment, a pharmaceutical composition is providedcomprising an anti-HIV effective amount of a population of human T cellsof a human having HIV/AIDS, and HIV-associated malignancy such aleukemia, leukemia of the CNS, sarcoma, Kaposi's sarcoma, or infectioussequelae associated with HIV/AIDS wherein the infection is refractory ornon-responsive to one or more highly active anti-retroviral therapies(ART) and/or chemotherapies that have failed to completely eradicate theHIV/AIDS or HIV-associated malignancy, respectively. The use ofCAR-modified T cells also includes the situation wherein the viralreservoir is reactivated or allowed to progress in order to decrease theratio of latently to actively infected cells including withdrawal of ART(therapeutic holiday), or active induction by agents that methylate ordemethylate promoter elements, activate cell activation pathways, ormimic epigenetic signals that activate latent virus to express theenvelope protein and thus become a CAR targets.

In another aspect, methods of making CAR-containing T cells (hereinafter“CAR-T cells”) are provided. The methods include transducing a T cellwith a vector or nucleic acid molecule encoding a disclosed CAR thatspecifically binds HIV envelope protein, thereby making the CAR-T cell.

In yet another aspect, a method of generating a population ofRNA-engineered cells is provided that comprises introducing an in vitrotranscribed RNA or synthetic RNA of a nucleic acid molecule encoding adisclosed CAR into a cell of a subject, thereby generating a CAR cell.

In yet another aspect, a method for diagnosing a disease, disorder orcondition associated with the expression of HIV envelope on a cell, isprovided comprising a) contacting the cell with a human anti-HIVantibody or fragment thereof, wherein the antibody or a fragment thereofcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96,100, 104, 112, 116, and 120; and b) detecting the presence of HIVenvelope wherein the presence of HIV envelope protein diagnoses for thedisease, disorder or condition associated with HIV/AIDS.

In one embodiment, the disease, disorder or condition associated withthe expression of HIV is any AIDS-defining illness, includingCandidiasis of the esophagus, bronchi, trachea, or lungs and mouth(thrush), Cervical cancer, invasive, Coccidioidomycosis, disseminated orextrapulmonary, Cryptococcosis, extrapulmonary, Cryptosporidiosis,chronic intestinal (greater than one month's duration), Cytomegalovirusdisease (other than liver, spleen, or nodes), Cytomegalovirus retinitis(with loss of vision), Encephalopathy, HIV related, Herpes simplex:chronic ulcer(s) (more than 1 month in duration); or bronchitis,pneumonitis, or esophagitis, Histoplasmosis, disseminated orextrapulmonary, Isosporiasis, chronic intestinal (more than 1 month induration), Kaposi sarcoma, Lymphoma, Burkitt's (or equivalent term,Lymphoma, immunoblastic (or equivalent term), Lymphoma, primary, ofbrain, Mycobacterium avium complex or M kansasii, disseminated orextrapulmonary, Mycobacterium tuberculosis, any site (pulmonary orextrapulmonary), Mycobacterium, other species or unidentified species,disseminated or extrapulmonary, Pneumocystis jiroveci pneumonia,Pneumonia, recurrent, Progressive multifocal leukoencephalopathy,Salmonella septicemia, recurrent, Toxoplasmosis of brain, Wastingsyndrome due to HIV (source: Revised classification system for HIVinfection and expanded surveillance case definition for AIDS amongadolescents and adults. Morbidity and Mortality Weekly Report, Dec. 18,1992/41 (RR-17), 1993). These may occur co-incident with cancerincluding hematopoietic cancer, myelodysplastic syndrome pancreaticcancer, head and neck cancer, cutaneous tumors, minimal residual disease(MRD) in ALL, AML, adult B cell malignancies including, CLL, CML, NHL,pediatric B cell malignancies (including B lineage ALL), multiplemyeloma lung cancer, breast cancer, ovarian cancer, prostate cancer,colon cancer, melanoma or other hematological cancer and solid tumors,or any combination thereof.

In another embodiment, a method of diagnosing, prognosing, ordetermining risk of an HIV-related disease in a mammal, is providedcomprising detecting the expression of HIV envelope in a sample derivedfrom the mammal comprising: a) contacting the sample with a humananti-HIV antibody or fragment thereof, wherein the antibody or afragment thereof comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76,80, 84, 88, 92, 96, 100, 104, 112, 116, and 120; and b) detecting thepresence of HIV wherein the presence of HIV diagnoses for an HIV-relateddisease in the mammal.

In another embodiment, a method of inhibiting HIV-dependent T cellinhibition, is provided comprising contacting a cell with a humananti-HIV antibody or fragment thereof, wherein the antibody or afragment thereof comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76,80, 84, 88, 92, 96, 100, 104, 112, 116, and 120. In one embodiment, thecell is selected from the group consisting of HIV-expressing cells,HIV-susceptible cells, and any combination thereof.

In another embodiment, a method of blocking T-cell inhibition mediatedby an HIV-expressing cell and altering infected tissues to inhibit HIVpathogenesis in a mammal, is provided comprising administering to themammal an effective amount of a composition comprising an isolatedanti-HIV antibody or fragment thereof, wherein the antibody or afragment thereof comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76,80, 84, 88, 92, 96, 100, 104, 112, 116, and 120. In one embodiment, thecell is selected from the group consisting of an HIV envelope expressingcell, HIV-infected cells or tissues, HIV-susceptible cells and tissues,and any combination thereof.

In another embodiment, a method of inhibiting, suppressing or preventingimmunosuppression of an anti-HIV response in a mammal, is providedcomprising administering to the mammal an effective amount of acomposition comprising an isolated anti-HIV antibody or fragmentthereof, wherein the antibody or a fragment thereof comprises an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6,46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116,and 120. In one embodiment, the antibody or fragment thereof inhibitsthe interaction between a first cell with a T cell, wherein the firstcell is selected from the group consisting of an HIV-expressing cell,HIV-infected cells or tissues, and any combination thereof.

In another aspect, a method is provided for inducing an anti-HIVimmunity in a mammal comprising administering to the mammal atherapeutically effective amount of a T cell transduced with vector ornucleic acid molecule encoding a disclosed CAR.

In another embodiment, a method of treating or preventing HIV infectionin a mammal is provided comprising administering to the mammal one ormore of the disclosed CARs, in an amount effective to treat or preventHIV infection in the mammal. The method includes administering to thesubject a therapeutically effective amount of host cells expressing adisclosed CAR that specifically binds HIV and/or one or more of theaforementioned antigens, under conditions sufficient to form an immunecomplex of the antigen binding domain on the CAR and the extracellulardomain of HIV and/or one or more of the aforementioned antigens in thesubject.

In yet another embodiment, a method is provided for treating a mammalhaving a disease, disorder or condition associated with an elevatedexpression of an HIV antigen, the method comprising administering to thesubject a pharmaceutical composition comprising an anti-HIV effectiveamount of a population of T cells, wherein the T cells comprise anucleic acid sequence that encodes a CAR, wherein the CAR includes atleast one extracellular HIV antigen binding domain comprising the aminoacid sequence of SEQ ID NOs. 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76,80, 84, 88, 92, 96, 100, 104, 112, 116, and 120 or any combinationthereof, at least one linker or spacer domain, at least onetransmembrane domain, at least one intracellular signaling domain, andwherein the T cells are T cells of the subject having cancer.

In yet another embodiment, a method is provided for treating cancer in asubject in need thereof comprising administering to the subject apharmaceutical composition comprising an anti-tumor effective amount ofa population of T cells, wherein the T cells comprise a nucleic acidsequence that encodes a CAR, wherein the CAR comprises at least one HIVantigen binding domain comprising the amino acid sequence of SEQ ID NOs.2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104,112, 116, and 120 or any combination thereof, at least one linker orspacer domain, at least one transmembrane domain, at least oneintracellular signaling domain, wherein the T cells are T cells of thesubject having HIV infection or T cells to be administered to such apatient. In some embodiments of the aforementioned methods, the at leastone transmembrane domain comprises a transmembrane the alpha, beta orzeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD8, CD9, CD16, CD19, CD22, Mesothelin, CD33, CD37, CD64, CD80, CD83,CD86, CD134, CD137, CD154, TNFRSF16, TNFRSF19, or a combination thereof.

In yet another embodiment, a method is provided for generating apersisting population of genetically engineered T cells in a humandiagnosed with HIV infection. In one embodiment, the method comprisesadministering to a human a T cell genetically engineered to express aCAR wherein the CAR comprises at least one HIV antigen binding domaincomprising the amino acid sequence of SEQ ID NOs. 2, 4, 6, 46, 50, 54,58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, and 120 orany combination thereof, at least one transmembrane domain, and at leastone intracellular signaling domain wherein the persisting population ofgenetically engineered T cells, or the population of progeny of the Tcells, persists in the human for at least one month, two months, threemonths, four months, five months, six months, seven months, eightmonths, nine months, ten months, eleven months, twelve months, twoyears, or three years after administration.

In one embodiment, the progeny T cells in the human comprise a memory Tcell. In another embodiment, the T cell is an autologous T cell.

In all of the aspects and embodiments of methods described herein, anyof the aforementioned infections, cancers, diseases, disorders orconditions associated with an elevated expression of a HIV antigen thatmay be treated or prevented or ameliorated using one or more of the CARsdisclosed herein.

In yet another aspect, each of the aforementioned bispecific and/ortrispecific anti-HIV CARs may be used in an adoptive T-cellimmunotherapy in an amount effective to either inhibit, suppress orprevent immunosuppression of an anti-HIV response in a mammal, or totreat, or prevent HIV infection in said mammal, wherein the mammalreceives the adoptive T-cell immunotherapy with either no prioranti-retroviral therapy (ART) treatment regimens being required, or toeffectively substantially reduce the number of ART treatment regimensrequired from approximately 10% to 99%.

In yet another aspect, a kit is provided for making a CAR T-cell asdescribed supra or for preventing, treating, or ameliorating any of theinfections, cancers, diseases, disorders or conditions associated withan elevated expression of a HIV antigen in a subject as described supra,comprising a container comprising any one of the nucleic acid molecules,vectors, host cells, or compositions disclosed supra or any combinationthereof, and instructions for using the kit.

While the disclosure provided herein has initially focused on thegeneration of CARs utilizing HIV envelope protein antigen bindingfragments thereof, it is predicted that the CARs, T Cell Receptors(TCRs) or nucleic acid sequences, polypeptides and methods of usethereof may be also employed with HIV protein other than the HIVenvelope protein antigen binding fragments specifically elucidatedherein, and are therefore meant to specifically include any HIV-derivedprotein associated with a latent or productive HIV infection, including,and not by way of limitation, Rev, Tat, Vif, Nef, Vpu, Vpr, Gag, Pol,Protease, Nucleocapsid, Matrix, Capsid, Integrase, and/or ReverseTranscriptase, or any combination thereof.

It will be understood that the CARs, host cells, nucleic acids, andmethods are useful beyond the specific aspects and embodiments that aredescribed in detail herein. The foregoing features and advantages of thedisclosure will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a schematic of the general domain structure of CARs withnovel extracellular HIV antigen binding domain sequences. MonospecificCAR-T (top construct) are composed of an extracellular HIV-bindingdomain (binder 1), a hinge and transmembrane domain (for example CD8hinge/spacer and CD8 transmembrane domain (TM), an intracellularsignaling CD137 costimulatory domain (41BB) and CD3zeta signalingdomain. Bispecific CAR-T (center three constructs) are composed of twobinders joined by a linker (black line) linked to hinge andtransmembrane domains and intracellular signaling domains (41BB,CD3zeta), or a single binder (binder 1) linked to a hinge/TM andintracellular signaling domains, followed by a 2A ribosomal skip site,then followed by a second binder (binder 2) linked to only ahinge/linker and transmembrane, or a hinge/linker transmembrane domainlinked to a second intracellular CD3zeta sequence. Trispecific CAR-T(bottom three constructs) are composed of either three linked anti-HIVbinders linked together to a single hinge/transmembrane sequence andintracellular signaling sequences (41BB and CD3zeta) or two binderslinked to each other and then to a hinge/transmembrane sequence followedby intracellular signaling motifs followed by a 2A ribosomal skip siteand then linked to either the third binder (binder 3) andhinge/transmembrane sequence only or hinge/transmembrane followed byCD3zeta intracellular signaling sequence.

FIGS. 2A-X depict several CARs containing novel extracellular HIVantigen binding domain sequences. The general scheme for the CARsincludes, from the N terminus to the C terminus, a Signal peptide,anti-HIV binder(s), extracellular linker, transmembrane, 4-1BB, CD3zeta. Bispecific and trispecific CAR constructs are also exemplified.

FIG. 2A depicts a lentiviral vector expressing the CAR containing theLTG1944 (LP-mD1.22-CD8TM-41BB-CD3zeta) nucleic acid sequence (SEQ ID NO:39) and the encoded amino acid sequence (SEQ ID NO: 40).

FIG. 2B depicts a lentiviral vector expressing the CAR containing theLTG1945 (LP-m36.4-CD8TM-41BB-CD3zeta) nucleic acid sequence (SEQ ID NO:41) and the encoded amino acid sequence (SEQ ID NO: 42).

FIG. 2C depicts a lentiviral vector expressing the CAR containing theLTG2328 (LP-C46-CD8TM-41BB-CD3zeta) nucleotide sequence (SEQ ID NO: 43)and the encoded amino acid sequence (SEQ ID NO: 44).

FIG. 2D depicts a lentiviral vector expressing the CAR containing theLTG2325 (LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta) nucleic acid sequence(SEQ ID NO: 47) and the encoded amino acid sequence (SEQ ID NO: 48).

FIG. 2E depicts a lentiviral vector expressing the CAR containing theLTG2313 (LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta) nucleic acid sequence(SEQ ID NO: 51) and the encoded amino acid sequence (SEQ ID NO: 52).

FIG. 2F depicts a lentiviral vector expressing the CAR containing theLTG1946 (LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta) nucleic acid sequence(SEQ ID NO: 55) and the encoded amino acid sequence (SEQ ID NO: 56).

FIG. 2G depicts a lentiviral vector expressing the CAR containing theLTG2326 (LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta) nucleic acid sequence(SEQ ID NO: 59) and the encoded amino acid sequence (SEQ ID NO: 60).

FIG. 2H depicts a lentiviral vector expressing the CAR containing theLTG1947 (LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta) nucleic acid sequence(SEQ ID NO: 63) and the encoded amino acid sequence (SEQ ID NO: 64).

FIG. 2I depicts a lentiviral vector expressing the CAR containing theLTG1948 (LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3zeta) nucleic acid sequence(SEQ ID NO: 67) and the encoded amino acid sequence (SEQ ID NO: 68).

FIG. 2J depicts a lentiviral vector expressing the CAR containing theLTG2303 (LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM-CD3zeta2)nucleic acid sequence (SEQ ID NO: 71) and the encoded amino acidsequence (SEQ ID NO: 72).

FIG. 2K depicts a lentiviral vector expressing the CAR containing theLTG2322 (LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM) nucleicacid sequence (SEQ ID NO: 73) and the encoded amino acid sequence (SEQID NO: 74).

FIG. 2L depicts a lentiviral vector expressing the CAR containing theLTG2314 (LP-mD1.22-L3-C46-CD8TM-41BB-CD3zeta) nucleic acid sequence (SEQID NO: 77) and the encoded amino acid sequence (SEQ ID NO: 78).

FIG. 2M depicts a lentiviral vector expressing the CAR containing theLTG2315 (LP-mD1.22-L5-C46-CD8TM-41BB-CD3zeta) nucleic acid sequence (SEQID NO: 81) and the encoded amino acid sequence (SEQ ID NO: 82).

FIG. 2N depicts a lentiviral vector expressing the CAR containing theLTG2316 (LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta) nucleic acid sequence (SEQID NO: 85) and the encoded amino acid sequence (SEQ ID NO: 86).

FIG. 2O depicts a lentiviral vector expressing the CAR containing theLTG2317 (LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta) nucleic acid sequence (SEQID NO: 89) and the encoded amino acid sequence (SEQ ID NO: 90).

FIG. 2P depicts a lentiviral vector expressing the CAR containing theLTG2318 (LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3zeta) nucleic acidsequence (SEQ ID NO: 93) and the encoded amino acid sequence (SEQ ID NO:94).

FIG. 2Q depicts a lentiviral vector expressing the CAR containing theLTG2319 (LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3zeta) nucleic acidsequence (SEQ ID NO: 97) and the encoded amino acid sequence (SEQ ID NO:98).

FIG. 2R depicts a lentiviral vector expressing the CAR containing theLTG2320 (LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta) nucleic acidsequence (SEQ ID NO: 101) and the encoded amino acid sequence (SEQ IDNO: 102).

FIG. 2S depicts a lentiviral vector expressing the CAR containing theLTG2323 (LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM)nucleic acid sequence (SEQ ID NO: 105) and the encoded amino acidsequence (SEQ ID NO: 106).

FIG. 2T depicts a lentiviral vector expressing the CAR containing theLTG2329(LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2) nucleic acid sequence (SEQ ID NO: 107) and the encoded amino acidsequence (SEQ ID NO: 108).

FIG. 2U depicts a lentiviral vector expressing the CAR containing theLTG2330(LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2) nucleic acid sequence (SEQ ID NO: 109) and the encoded amino acidsequence (SEQ ID NO: 110).

FIG. 2V depicts a lentiviral vector expressing the CAR containing theLTG2331 (LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM)nucleic acid sequence (SEQ ID NO: 113) and the encoded amino acidsequence (SEQ ID NO: 114).

FIG. 2W depicts a lentiviral vector expressing the CAR containing theLTG2332 (LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-m36.4-TNFRSF19TM)nucleic acid sequence (SEQ ID NO: 117) and the encoded amino acidsequence (SEQ ID NO: 118).

FIG. 2X depicts a lentiviral vector expressing the CAR containing theLTG2334 (LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TM)nucleic acid sequence (SEQ ID NO: 121) and the encoded amino acidsequence (SEQ ID NO: 122).

FIGS. 3A-E depict the functional characterization of monospecificanti-HIV CARs.

FIG. 3A shows expression of anti-HIV CARs containing either the mD1.22domain or C46 peptide on the surface of LV transduced human T cells. ThemD1.22-CAR is detected by staining with anti-CD8 antibody (y-axis) andanti-CD4 antibody (recognizes the mD1.22 domain, x-axis left panel) orthe C46-CAR is detected by staining with the 2F5 antibody (recognizesthe C46 peptide, x-axis, right panel).

FIG. 3B depicts how m36.4 expression was detected on the surface of LVtransduced primary T-cells by fusing the mCherry reporter directlyupstream of the CD3 zeta signaling domain. The % of mCherry positivecells were detected in the total T-cell population (left panel), on thesurface of CD4⁺ T-cells (middle panel) and CD8⁺ T-cells (right panel).

FIG. 3C demonstrates the differential cytotoxic function of monospecificCARs. Using an HIV-envelope expressing target cell line as acytotoxicity target, untransduced T cells (UTD, no activity), T cellstransduced with LTG1732 (control mCherry vector, no activity), LTG1944(expresses the mD1.22 binder), LTG1945 (expresses the m36.4 binder),LTG2328 (expresses the C46 binder) each demonstrated cytotoxicity at theeffector to target cell ratios (E:T) listed on the x-axis.

FIG. 3D demonstrates that the same transduced T cell populations doesnot mediate cytolytic activity against a cell line that does not expressthe HIV envelope protein.

FIG. 3E demonstrates that human T cells transduced with LV expressingmonospecific CARs secrete IFN-γ in the presence (solid bars) of HIVenvelope-expressing cells. Background levels are seen with cells notexpressing HIV envelope (checkered bars) or cultured alone (stripedbars).

FIGS. 4A-E depict the expression of bispecific anti-HIV CARs on thesurface of activated T-cells. Bispecific CARs are engineered in severalunique configurations to determine the precise functional architecture.

FIG. 4A depicts the bispecific anti-HIV CARs that are engineered withtwo anti-gp120 binders (mD1.22 and m36.4) and fused together using aflexible glycine-serine linker consisting of up to five GGGGS (G4S)motifs. The bispecific CARs are detected by flow cytometry using anantibody directed against the D1 domain of the CD4 receptor (anti-CD4VIT4 clone, Miltenyi Biotec) which also recognizes the mD1.22 domain ofthe bispecific CARs.

FIG. 4B depicts the expression of a bispecific CAR engineered with them36.4 domain presented as the first gp120 binder and then fused to themD1.22 domain using three G4S motifs (LTG1948).

FIG. 4C depicts the expression of a bispecific CAR with mD1.22 and m36.4domains fused to their own intracellular CD3 zeta chains via abicistronic construct (LTG2303) incorporating the porcine teschovirus-1derived self-cleaving P2A peptide. The expression of the LTG2303 CAR onthe surface of T-cells was determined by detecting the mD1.22 domainusing anti-CD4 flow cytometry.

FIG. 4D depicts the western blot detection of both mD1.22-CAR andm36.4-CAR using anti-CD3 zeta staining. Both CARs are fully cleavedusing the P2A self-cleaving peptide. Endogenous CD3 zeta serves as aloading control.

FIG. 4E depicts the graphical representation of % of CAR modifiedT-cells via detection of the mD1.22 by anti-CD4 flow cytometry.

FIG. 5 depicts the potent ability of anti-HIV CARs to destroyHIV-envelope expressing 293T cells that also co-express the fireflyluciferase reporter. Notably, the bispecific anti-HIV CAR configured sothat each anti-HIV binder is on a single CD3 zeta chain (LTG2303)significantly killed target cells as compared to monospecific CARs,LTG1944 and LTG1945. Untransduced T-cells (no activity) and LTG1732(T-cells transduced with the mCherry reporter, no activity) served asnegative controls to monitor non-specific T-cell mediated cytotoxicity.The results shown are from three donors. Error bars represent+/−standard deviation. Statistical analysis was performed by two-wayANOVA (***P<0.0001, **P<0.001, *P<0.01).

FIG. 6 depicts the exceptional specificity of anti-HIV CARs in theabsence of HIV envelope expressing cell lines. Raji cells are known toexpress MHC class II molecules and thus may interact non-specificallywith the CD4 receptor derived mD1.22 domain. As demonstrated on bothcell lines, no off-target cytolysis was observed for anti-HIV CARs.Representative figure of three donors. The error bars represent+/−standard deviation.

FIG. 7 shows that activation of anti-HIV CAR-T triggers IFN-gammarelease. Anti-HIV CARs were co-incubated with (Env⁺) or without (Env⁻)envelope-expressing 293T cells that also co-express the fireflyluciferase reporter. Twenty-four hours later, cell culture supernatantswere collected and assayed for IFN-γ using ELISA. All anti-HIV CARs wereactivated in the presence of Env⁺ cells and to a lesser extent in thepresence of Env⁻ cells. Bispecific CAR LTG2303 demonstrated enhancedIFN-gamma secretion over that of monospecific CARs, LTG1944 and LTG1945.Results shown are from two different donors. The error bars represent+/−standard deviation.

FIGS. 8A-B show that bispecific anti-HIV CAR function is governed bylinker length.

FIG. 8A depicts bispecific CARs were constructed with up to five G4Smotifs and their function investigated for their ability to destroyenvelope-expressing target cells (Env⁺).

FIG. 8B depicts the specificity of bispecific anti-HIV CARs in thepresence of envelope-negative Raji cells (Env⁻). The bispecific CAR witha single G4S motif was significantly more active than an anti-HIV CARcontaining five G4S motifs. Statistics was performed by two-way ANOVA(***p<0.0001, LTG2325 vs. LTG1947). The error bars represent +/−standarddeviation.

FIG. 9 shows that bispecific anti-HIV CARs engineered with increasinglinker lengths are triggered to release IFN-gamma in the presence ofenvelope-expressing target cells (Env⁺) and to a lesser extent in theabsence of envelope-expressing cells (Env⁻) or when effectors arecultured alone. The anti-HIV CAR with the longest linker (LTG1947)releases the lowest background level of IFN-gamma. The error barsrepresent +/−standard deviation.

FIGS. 10A-B show that the bispecific anti-HIV CARs engineered with themD1.22 and C46 peptide exhibit configuration-dependent activity.

FIG. 10A depicts the cytotoxic activity of C46-based bispecific CARs.The cytotoxicity of these CARs depends highly on the configuration ofthe bispecific gp120/gp41-binder. For example, when the C46 peptide isoriented distal to the mD1.22 domain to form CARs, LTG2316 and LTG2317,both CARs function similar to the LTG1946 CAR containing both the mD1.22and m36.4 domains. In contrast, orienting mD1.22 distal to the C46peptide to form CARs, LTG2314 and LTG2315, completely abolished itscytolytic function.

FIG. 10B demonstrates that C46-based bispecific CARs elaborate IFN-gammain the presence of Env⁺ target cells and in some instancesnon-specifically in its absence (Env⁻ or effectors alone).

FIGS. 11A-F show that trispecific anti-HIV CARs robustly destroy andrelease cytokines in response to envelope-expressing target cells.

FIG. 11A depicts the expression of trispecific anti-HIV CARs on thesurface of primary T-cells using a broadly-neutralizing 2F5 antibodydirected against the C46 peptide. The 2F5 antibody recognizes acontiguous epitope within the C46 peptide (ELDKWA).

FIG. 11B depicts the detection of the mD1.22 domain found within thebispecific arm of the trispecific anti-HIV CAR, LTG2323. The mD1.22domain was detected on the surface of T-cells using anti-CD4 (recognizesthe mD1.22 domain) and anti-CD8 flow cytometry. This construct containsa bispecific CAR (LTG1946) in combination with a membrane-anchored C46peptide presented on the surface of T-cells as a discrete entity.

FIG. 11C depicts the robust destruction of HIV-envelope (Env⁺)expressing cells by all trispecific CARs.

FIG. 11D depicts the specificity of the trispecific CARs in the absenceof envelope expressing but MHC class II expressing Raji cells (Env⁻).

FIG. 11E depicts the release of IL-2 cytokine for all trispecific CARsas compared to the highly potent LTG1946 bispecific CAR.

FIG. 11F depicts the release of IFN-γ cytokine for all trispecific CARsas compared to the highly potent LTG1946 bispecific CAR.

FIGS. 12A-D show a comparison of the expression and cytotoxicity exertedby the most potent trispecific and bispecific anti-HIV CARs.

FIG. 12A depicts percentage of bispecific CAR-modified T-cells viadetection of the mD1.22 domain using anti-CD4 flow cytometry.

FIG. 12B depicts the cytotoxicity of trispecific anti-HIV CARs.

FIG. 12C depicts the percentage of trispecific CAR-modified T-cells viadetection of the C46 domain using 2F5 flow cytometry. The dotted lineintersecting the y-axis is set for 30% gene modification.

FIG. 12D depicts the specificity of trispecific anti-HIV CARs asmeasured by cytotoxicity on an envelope-negative cell line. Experimentaldata shown in this figure are of at least 3 donors (error bars=+/−S.D.).

FIG. 13 shows the CD4⁺ and CD8⁺ composition of donors used in the invitro and in vivo HIV-1 challenge studies. Donors were enriched for CD4⁺and CD8⁺ effector T cells, activated, and expanded for up to 9 days. Onday 9, the composition of donor T cells was determined by anti-CD4 andanti-CD8 flow cytometry as described in Example 1. This figure shows thepercentage of CD4⁺ and CD8⁺ effectors on day 9 of culture.

FIGS. 14A-K show the in vitro killing efficacy of monospecific,bispecific, and trispecific anti-HIV CAR-T cells against PBMC infectedwith replication-competent, infectious molecular clones of HIV-1encoding broad envelope (env) genes and the Renilla luciferase reporter(Env-IMC-LucR). The indicated anti-HIV CAR or untransduced (UTD) T cells(effectors) were co-cultured with autologous PBMC infected with theindicated Env-IMC-LucR virus (targets) one day prior to co-culturesetup. Seven days later, the co-cultures were lysed and luciferaseactivity quantified. The bispecific and trispecific anti-HIV duoCAR-Tcells eliminate PBMC infected with broad Env-IMC-LucR viruses (LTG2303,LTG2329, and LTG2330) across different donors.

FIG. 14A shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR virus encoding a laboratory-adapted NL4-3 env gene (cladeB, X4-tropic).

FIG. 14B shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR virus encoding a clade B HIV-1 env gene isolated from theUSA (BaL, R5-tropic).

FIG. 14C shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR virus encoding a second clade B HIV-1 env gene isolatedfrom the USA (SF162, R5-tropic).

FIG. 14D shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR virus encoding a VRC01 and 3BNC117-resistant clade C HIV-1env gene isolated from S. Africa (C.Du422.1).

FIG. 14E shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR virus encoding a VRC01-resistant clade C HIV-1 env geneisolated from S. Africa (C.Du172.17).

FIG. 14F shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR virus encoding a VRC01 partially-resistant clade C HIV-1env gene isolated from S.E. Africa (C. Cap45).

FIG. 14G shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR virus encoding a representative clade AC HIV-1 env geneisolated from E. Africa (AC.246-F3.LucR).

FIG. 14H shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR encoding a representative clade BC HIV-1 env gene isolatedfrom China (BC.CH119.10.LucR).

FIG. 14I shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR virus encoding a representative clade G HIV-1 env geneisolated from Spain (GX1632_S2_B10.LucR).

FIG. 14J shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR virus encoding a representative clade AE HIV-1 env genefound in S. China and/or Thailand (AE.CNE8.LucR).

FIG. 14K shows CAR-T killing efficacy against PBMC infected with anEnv-IMC-LucR virus encoding a second representative clade AE HIV-1 envgene found in S. China and/or Thailand (AE.CNE55.LucR). The x-axis islabeled with the indicated anti-HIV CAR and “T” represents “target” orEnv-IMC-LucR-infected PBMCs. Error bars represent +/−S.D. Statisticalanalysis was performed by multiple comparison student's t-test.Significance of findings are indicated on the graph.

FIG. 15 shows a summary of the in vitro CAR-T killing efficacy for alldonors tested expressed as log inhibition of HIV-1 infection. Loginhibition is calculated relative to HIV-infected untransduced T cellsafter background subtraction using uninfected PBMCs. The data shows anaverage of at least three independent donors.

FIG. 16 shows a summary of the in vitro CAR-T killing efficacy for alldonors tested expressed as % inhibition of HIV-1 infection. Percentinhibition is calculated relative to HIV-infected untransduced T cellsafter background subtraction using uninfected PBMCs. The data shows anaverage of at least three independent donors.

FIG. 17A-B shows that multi-specific anti-HIV duoCAR T cells exhibitsuperior in vitro killing efficacy at very low E:T ratios.

FIG. 17A depicts donor matched PBMC infected with Du422.1-IMC-LucR viruson Day −1 (T=targets) followed by addition of the indicated anti-HIVCAR-T cells (E=effectors) on Day 0 at different E:T ratios (1:1, 0.5:1,0.25:1, and 0.125:1).

FIG. 17B depicts donor matched PBMC infected with Du422.1-IMC-LucR viruson Day −1 followed by addition of the indicated anti-HIV CAR-T cells onDay 0 at different E:T ratios (1:25, 1:50, and 1:100). Seven dayspost-addition of CAR-T cells, the co-cultures were harvested and assayedfor Renilla luciferase activity. Statistical analysis was performed bymultiple comparison student's t test. Statistical significance isconsidered P<0.05 and was determined by the Holm-Sidak method.P<0.00001****, P<0.0001***, P<0.001**, P<0.05*.

FIGS. 18A-J show broad in vitro protection of anti-HIV CAR-T cells forindividual donors. For some donors, the mD1.22-CAR-T cells are moresusceptible to HIV-1 infection. Conversely, the m36.4 domain protectsthe bispecific and trispecific CAR T cells from HIV-1 infectionindependent of its architecture.

FIG. 18A shows in vitro protection of anti-HIV CAR-T cells challengedwith Env-IMC-LucR virus encoding the NL4-3 env gene.

FIG. 18B shows in vitro protection of anti-HIV CAR-T cells challengedwith Env-IMC-LucR virus encoding the BaL env gene.

FIG. 18C shows in vitro protection of anti-HIV CAR-T cells challengedwith Env-IMC-LucR virus encoding the C.Du422.1 env gene.

FIG. 18D shows in vitro protection of anti-HIV CAR-T cells challengedwith Env-IMC-LucR virus encoding the C.Du172.17 env gene.

FIG. 18E shows in vitro protection of anti-HIV CAR-T cells challengedwith Env-IMC-LucR virus encoding the AC.246-F3 env gene (clade AC).

FIG. 18F shows in vitro protection of anti-HIV CAR-T cells challengedwith Env-IMC-LucR virus encoding a representative clade BC env gene(BC.CH119.10).

FIG. 18G shows in vitro protection of anti-HIV CAR-T cells challengedwith Env-IMC-LucR virus encoding a representative clade AE env gene(AE.CNE8).

FIG. 18H shows in vitro protection of anti-HIV CAR-T cells challengedwith Env-IMC-LucR virus encoding a representative clade G env gene(GX1632_S2_B10).

FIG. 18I shows in vitro protection of anti-HIV CAR-T cells challengedwith Env-IMC-LucR virus encoding a second representative clade AE envgene (AE.CNE55).

FIG. 18J shows all donors plotted together for each Env-IMC-LucR virustested. The error bars represent +/−S.D. Statistical analysis wasperformed by two-way ANOVA followed by Bonferroni post-test. Significantfindings are indicated on the graph.

FIGS. 19A-I shows that bispecific and trispecific anti-HIV duoCAR-Tcells potently eliminate PBMC infected with bNAb-resistant virus(Du422.1-IMC-LucR) in vivo.

FIG. 19A depicts an illustration of the humanized intrasplenic PBMC NSGacute and chronic HIV-1 infection model (hu-spl-PBMC-NSG).

FIG. 19B depicts the in vivo killing efficacy of bispecific andtrispecific anti-HIV duoCAR-T cells against PBMC infected withbNAb-resistant Du422.1-IMC-LucR virus for 7 days (acute).

FIG. 19C depicts the in vivo killing efficacy of bispecific andtrispecific anti-HIV duoCAR-T cells against PBMC infected withbNAb-resistant Du422.1-IMC-LucR virus for 30 days (chronic). Data showsluciferase activity detected in the spleens of mice at day 7 (acute) orday 30 (chronic). Day 30 luciferase activity (RLU) was normalized to %CD4⁺ T cells to account for loss of CD4⁺ T cells in the UTD controlgroup due to uncontrolled HIV-1 infection.

FIG. 19D depicts the % of CD4⁺ T cells isolated from mice spleens at day7 (acute).

FIG. 19E depicts the % of CD4⁺ T cells isolated from mice spleens at day30 (chronic).

FIG. 19F depicts the % of CD8⁺ isolated from the mice spleens during day7 (acute).

FIG. 19G depicts the % of CD8⁺ isolated from the mice spleens during day30 (chronic).

FIG. 19H depicts the CAR copies/μg of splenic DNA at day 7 (acute).

FIG. 19I depicts the CAR copies/μg of splenic DNA at day 30 (chronic).Statistical analysis was performed by one-way ANOVA followed by tukey'spost-test. Significance of findings are indicated on the graph.

DETAILED DESCRIPTION Definitions

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” Thus,“comprising an antigen” means “including an antigen” without excludingother elements. The phrase “and/or” means “and” or “or.” It is furtherto be understood that any and all base sizes or amino acid sizes, andall molecular weight or molecular mass values, given for nucleic acidsor polypeptides are approximate, and are provided for descriptivepurposes, unless otherwise indicated. Although many methods andmaterials similar or equivalent to those described herein can be used,particular suitable methods and materials are described below. In caseof conflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting. To facilitate reviewof the various embodiments, the following explanations of terms areprovided:

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations of.+−0.20% or in some instances .+−0.10%, or in some instances .+−0.5%, orin some instances .+−0.1%, or in some instances .+−0.0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods.

Unless otherwise noted, the technical terms herein are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 1999; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995; and other similarreferences.

The present disclosure provides for HIV antibodies or fragments thereofas well as CARs having such HIV antigen binding domains. The enhancementof the functional activity of the CAR directly relates to theenhancement of functional activity of the CAR-expressing T cell. As aresult of one or more of these modifications, the CARs exhibit both ahigh degree of cytokine-induced cytolysis and cell surface expression ontransduced T cells, along with an increased level of in vivo T cellexpansion and persistence of the transduced CAR-expressing T cell.

The unique ability to combine functional moieties derived from differentprotein domains has been a key innovative feature of CARs. The choice ofeach of these protein domains is a key design feature, as is the way inwhich they are specifically combined. Each design domain is an essentialcomponent that can be used across different CAR platforms to engineerthe function of lymphocytes. For example, the choice of theextracellular binding domain can make an otherwise ineffective CAR beeffective.

The invariable framework components of the immunoglobulin-derivedprotein sequences used to create the extracellular antigen bindingdomain of a CAR can either be entirely neutral, or they canself-associate and drive the T cell to a state of metabolic exhaustion,thus making the therapeutic T cell expressing that CAR far lesseffective. This occurs independently of the antigen binding function ofthis CAR domain. Furthermore, the choice of the intracellular signalingdomain(s) also can govern the activity and the durability of thetherapeutic lymphocyte population used for immunotherapy. While theability to bind target antigen and the ability to transmit an activationsignal to the T cell through these extracellular and intracellulardomains, respectively, are important CAR design aspects, what has alsobecome apparent is that the choice of the source of the extracellularantigen binding fragments can have a significant effect on the efficacyof the CAR and thereby have a defining role for the function andclinical utility of the CAR.

Surprisingly and unexpectedly it has now been discovered that use of anentirely human antigen binding domain in a CAR, rather than usingmouse-derived antigen binding fragments which are prone to induceanti-mouse immune response and CAR T elimination in a host (c.f., theUPenn-sponsored clinical trial using mouse derived SS1 ScFv sequence,NCT02159716), may also determine the functional activity of aCAR-expressing T cell.

The CARs disclosed herein are expressed at a high level in a cell. Acell expressing the CAR has a high in vivo proliferation rate, produceslarge amounts of cytokines and has a high cytotoxic activity against acell having, on its surface, HIV envelope antigen to which a CAR binds.The use of a human extracellular HIV antigen binding domain results ingeneration of a CAR that functions better in vivo, while avoiding theinduction of anti-CAR immunity in the host immune response and thekilling of the CAR T cell population. The CARs expressing the entirelyhuman extracellular HIV antigen binding domains exhibit superioractivities/properties including i) prevention of poor CAR T persistenceand function as seen with mouse-derived binding sequences; ii) lack ofregional (i.e. intrapleural) delivery of the CAR to be efficacious; andiii) ability to generate CAR T cell designs based both on binders withhigh and low affinity to HIV.

What follows is a detailed description of the inventive CARs including adescription of their extracellular HIV antigen binding domain, thetransmembrane domain and the intracellular domain, along with additionaldescription of the CARs, antibodies and antigen binding fragmentsthereof, conjugates, nucleotides, expression, vectors, and host cells,methods of treatment, compositions, and kits employing the disclosedCARs.

A. Chimeric Antigen Receptors (CARs)

The CARs disclosed herein comprise at least one HIV antigen bindingdomain capable of binding to HIV envelope protein, at least onetransmembrane domain, and at least one intracellular domain.

A chimeric antigen receptor (CAR) is an artificially constructed hybridprotein or polypeptide containing the antigen binding domains of anantibody or a receptor (e.g., single chain variable fragment (ScFv), orhuman CD4 which binds to the gp120 portion of HIV envelope protein)linked to T-cell signaling domains via the transmembrane domain via thelinker or hinge domain. Characteristics of CARs include their ability toredirect T-cell specificity and reactivity toward a selected target in anon-Major Histocompatibility Complex (MHC)-restricted manner, andexploiting the antigen-binding properties of monoclonal antibodies orreceptors. The non-MHC-restricted antigen recognition gives T cellsexpressing CARs the ability to recognize antigen independent of antigenprocessing. Moreover, when expressed in T-cells, CARs advantageously donot dimerize with endogenous T cell receptor (TCR) alpha and betachains.

As disclosed herein, the intracellular T cell signaling domains of theCARs can include, for example, a T cell receptor signaling domain, a Tcell costimulatory signaling domain, or both. The T cell receptorsignaling domain refers to a portion of the CAR comprising theintracellular domain of a T cell receptor, such as, for example, and notby way of limitation, the intracellular portion of the CD3 zeta protein.The costimulatory signaling domain refers to a portion of the CARcomprising the intracellular domain of a costimulatory molecule, whichis a cell surface molecule other than an antigen receptor or theirligands that are required for an efficient response of lymphocytes toantigen.

1. Extracellular Domain

In one embodiment, the CAR comprises a target-specific binding elementotherwise referred to as an antigen binding domain or moiety. The choiceof domain depends upon the type and number of ligands that define thesurface of a target cell. For example, the antigen binding domain may bechosen to recognize a ligand that acts as a cell surface marker ontarget cells associated with a particular disease state. Thus examplesof cell surface markers that may act as ligands for the antigen bindingdomain in the CAR include those associated with viral, bacterial andparasitic infections, autoimmune disease and cancer cells.

In one embodiment, the CAR can be engineered to target a viral antigenof interest by way of engineering a desired antigen binding domain thatspecifically binds to an antigen on a virus infected cell. Viralantigens are proteins that are produced by virus infected cells thatelicit an immune response, particularly T-cell mediated immuneresponses. The selection of the antigen binding domain will depend onthe particular type of virus infection to be treated. Viral antigensthat can serve as CAR targets include those expressed on the surface ofan infected cell such as, HIV envelope protein glycoprotein (gp160,gp120/gp41), Hepatitis B Virus (HBV) surface antigen (HBsAg), HepatitisC virus (HBV) E2 glycoprotein, Epstein-Barr envelope protein, andcytomegalovirus (CMV) glycoprotein B antigen. The viral antigensdisclosed herein are merely included by way of example. The list is notintended to be exclusive and further examples will be readily apparentto those of skill in the art.

In one embodiment, the HIV envelope protein comprises one or moreantigenic epitopes associated with the viral envelope protein. HIVinfected cells express envelope protein that can serve as target antigenfor an immune attack. In this disclosure, anti-HIV CARs have beendeveloped that target the HIV envelope protein using a singleextracellular domain to form a monospecific CAR; combination of two ofthese extracellular domains in different orientations and with varyinglinker lengths to form a bispecific CAR; or a combination of all threeextracellular domains to form a trispecific CAR. The CARs are composedof three functionally distinct extracellular domains that targetnon-overlapping epitopes on the HIV envelope to effectively block HIVentry (mD1.22), coreceptor usage (m36.4), and viral fusion (C46). Incontrast to CARs containing either a single bnAb and/or the wild-typeCD4 receptor, all three extracellular domains have been preciselyengineered to be compact with enhanced specificity, potency, and theability to target newly emerging T20-resistant strains.

The mD1.22 domain is derived from the human CD4 receptor and targets ahighly conserved epitope on HIV-1 gp120. In comparison to thefull-length CD4 receptor, mD1.22 has been engineered to be molecularlycompact, with high specificity, affinity, and potency against diverseHIV-1 clades (Chen et al., J. Virol. 2014; 88:(2) 1125-1139). The m36.4domain is an affinity-matured, engineered human antibody domain composedof the heavy chain only (Chen et al., Antiviral Research 2010; 88:(1)107-115). It binds to a discontinuous CD4-induced (CD4i) epitope ongp120 in the vicinity of the coreceptor binding site (Wan et al. PLOSOne 2013; 8(6):e66638). When combined together, the two domains worksynergistically to potently neutralize HIV-1 and inhibit viral entry(Chen et al., J. Virol. 2014; 88:(2) 1125-1139).

Targeting multiple non-overlapping epitopes is an attractive strategy toprevent viral escape. As a third layer of T-cell protection, CAR-T orT-cells themselves were engineered with the highly potent C46 fusioninhibitor. The C46 peptide belongs to a class of gp41-derived“C-peptide” fusion inhibitors that block HIV-1 infection at the level ofviral fusion. It is an extended version of the FDA-approved enfuviritideor T20 and similar to T20, the C46 peptide can be used to suppress HIVvariants with multidrug resistance to current cART. Notably whenC-peptides are expressed on the surface of T-cells or secreted fromT-cells (SAVE peptides), they potently abrogate HIV-1 fusion to theT-cell membrane (van Lunzen et al., Molecular Therapy 2007, 15: (5)1024-1033; Kimpel et al., PLOS One 2010, 5:(8) e12357; Egerer et al.,Molecular Therapy 2010, 19(7), 1236-1244). Hence, CARs engineered withthese three domains are designed to potently destroy HIV-infected cellswhile conferring protection to the CAR T-cell.

To reduce immunogenicity, the CARs employed here are constructed offully human sequences. This is an advantage over using mouse-based ScFvbinding sequences, that are prone to induce immune response and CAR-Telimination in a human host leading to poor T-cell persistence. Asdescribed herein, CAR transduced T-cells have been generated bytransduction with a lentiviral vector construct encoding the anti-HIVCAR gene. It is expected that the CAR-expressing T-cells will havelong-lasting therapeutic effect in the patient. It is important to notethat the therapeutic CAR-T cell product will be given once rather thanthrough a series of recurring IV administrations.

In general, mD1.22-based CARs (containing a minimal domain human CD4protein fragment) are highly potent and specifically destroyed 293Tcells stably expressing HIV gp120 (referred to as, env⁺ cells), whichserves as a surrogate HIV-infected cell, and used to reproduciblyquantify CAR-mediated cytotoxicity. Subsequently fusing the mD1.22domain to m36.4 (an anti-HIV antibody derived binding domain) usinglinker domains, as in SEQ ID NOs: 24, 26, 28, 30, 32 and then joiningthese domains to CD8 or to TNFRSF19 transmembrane domains (TM), SEQ IDNO: 8 and SEQ ID NO: 14, by virtue of a linker domain derived from CD8,as in SEQ ID NO: 10; resulting in a CD8 linker joined to a CD8transmembrane domain as in SEQ ID NO: 12; or CD8 linker linked toTNFRSF19TM as in SEQ ID no: 16; thus allowing for linkage tointracellular signaling domains. The intracellular signaling domains areeither first generation or second generation. The second generation CARslink the 41BB (CD137) intracellular signaling domain, SEQ ID NO: 18,proximal to the transmembrane followed by CD3zeta signaling domain. Theintracellular domains for CD3 zeta chain are linked directly to thetransmembrane domain or in second generation constructs to the 41BBdomain. In the creation of lentiviral gene vectors it is crucial toavoid sequence repetition as the vectors often edit or recombine outthese sequences, and the structure of the CAR construct is then lost.Another unique characteristic of the vector family presented here is thecreation of two different CD3 zeta domains. They both code for the sameamino acid sequence, SEQ ID NO: 20 and SEQ ID NO: 22, yet diverge intheir nucleic acid sequence, SEQ ID NO: 19 and SEQ ID NO: 21.

In some cases, a single chain forms a bispecific or trispecific CAR,that contains two or three extracellular domains linked and expressed onthe same final cell surface protein. In other cases, two separate chainsare expressed, being encoded in the same construct however, processedinto two proteins by including a composite furin/ribosomal skip site orself-cleaving peptide, as in SEQ ID NO: 33 and SEQ ID NO: 34. Theimportance of a bispecific or trispecific CAR should be realized in theevent that one binder is unable to recognize a HIV variant or an evolvedmutation, the second and/or third domain may compensate for the loss.The effective use of these CAR constructs described herein willeliminate ART dependency and move towards a cure for HIV using CAR-Ttechnology.

In one preferred embodiment, the target antigen is the HIV envelopeprotein and the infected cells and tissues associated with expression ofHIV envelope that comprise and essentially define the status of beingHIV infected, including infected epithelial tissues, lymphoid tissues,and lymphocytes.

In a preferred embodiment, the antigen binding domain portion of the CARtargets the extracellular HIV envelope antigen.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe extracellular HIV envelope antigen binding domain comprises anucleotide sequence of SEQ ID NO: 1, or a sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity thereof. In one embodiment, an isolatednucleic acid molecule is provided wherein the encoded extracellular HIVantigen binding domain comprises an amino acid sequence of SEQ ID NO: 2,or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to an amino acid sequence of SEQ ID NO: 2.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe extracellular HIV envelope antigen binding domain comprises anucleotide sequence of SEQ ID NO: 3, or a sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity thereof. In one embodiment, an isolatednucleic acid molecule is provided wherein the encoded extracellular HIVantigen binding domain comprises an amino acid sequence of SEQ ID NO: 4,or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to an amino acid sequence of SEQ ID NO: 4.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe extracellular HIV envelope antigen binding domain comprises anucleotide sequence of SEQ ID NO: 5, or a sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity thereof. In one embodiment, an isolatednucleic acid molecule is provided wherein the encoded extracellular HIVantigen binding domain comprises an amino acid sequence of SEQ ID NO: 6,or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to an amino acid sequence of SEQ ID NO: 6.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 45, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 46, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:46.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 49, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 50, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:50.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 53, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 54, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:54.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 57, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 58, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:58.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 61, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 62, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:62.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 65, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 66, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:66.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 69, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 70, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:70.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 75, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 76, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:76.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 79, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 80, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:80.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 83, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 84, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:84.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domain comprises anucleotide sequence of SEQ ID NO: 87, or a sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity thereof. In one embodiment, an isolatednucleic acid molecule is provided wherein the encoded extracellular HIVantigen binding domain comprises an amino acid sequence of SEQ ID NO:88, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99%identity to an amino acid sequence of SEQ ID NO: 88.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 91, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 92, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:92.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 95, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 96, or an amino acid sequence with 85%, 90%, 95%,96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO:96.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 99, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 100, or an amino acid sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ IDNO: 100.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 103, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 104, or an amino acid sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ IDNO: 104.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 111, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 112, or an amino acid sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ IDNO: 112.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 115, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 116, or an amino acid sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ IDNO: 116.

In one preferred embodiment, the isolated nucleic acid molecule encodingthe linked extracellular HIV envelope antigen binding domains comprisesa nucleotide sequence of SEQ ID NO: 119, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof. In one embodiment, anisolated nucleic acid molecule is provided wherein the encodedextracellular HIV antigen binding domain comprises an amino acidsequence of SEQ ID NO: 120, or an amino acid sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ IDNO: 120.

In the various embodiments of the HIV-specific CARs disclosed herein,the general scheme is set forth in FIG. 1 and includes, from theN-terminus to the C-terminus, a signal or leader peptide, anti-HIVbinders, extracellular linker, CD8 transmembrane, 4-1BB, and CD3zeta.Also shown are CARs that incorporate multiple binders (bispecific,trispecific) joined by specific linkage domains.

In one embodiment, the nucleic acid sequence encoding a CAR comprisesthe nucleic acid sequence of SEQ ID NO: 39, and encodes the CARcomprising the amino acid sequence as set forth in SEQ ID NO: 40[LTG1944, LP-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (as depictedin FIG. 2A)].

In one embodiment, the nucleic acid sequence encoding a CAR comprisesthe nucleic acid sequence of SEQ ID NO: 39, or a sequence with 85%, 90%,95%, 96%, 97%, 98% or 99% identity thereof, and encodes the CARcomprising the amino acid sequence as set forth in SEQ ID NO: 40 or asequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof,[LTG1944, LP-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (as depictedin FIG. 2A)].

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 41, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 42[LTG1945, LP-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (as depictedin FIG. 2B)].

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 41 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 42 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG1945, LP-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (as depictedin FIG. 2B)].

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 43, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 44[LTG2328, LP-C46-CD8TM-41BB-CD3zeta amino acid sequence (as depicted inFIG. 2C)].

In another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 43 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 44 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2328, LP-C46-CD8TM-41BB-CD3zeta amino acid sequence (as depicted inFIG. 2C)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 47, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 48[LTG2325, LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2D)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 47 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 48 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2325, LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2D)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 51, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 52[LTG2313, LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2E)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 51 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 52 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2313, LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2E)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 55, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 56[LTG1946, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2F)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 55 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 56 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG1946, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2F)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 59, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 60[LTG2326, LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2G)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 59 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 60 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2326, LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2G)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 63, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 64[LTG1947, LP-mD1.22-L5-m36.4-CD8 TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2H)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 63 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 64 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG1947, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2H)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 67, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 68[LTG1948, LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2I)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 67 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 68 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG1948, LP-m36.4-L3-mD1.22-CD8 TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2I)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 71, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 72[LTG2303,LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2 aminoacid sequence (as depicted in FIG. 2J)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 71 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 72 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2303,LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2 aminoacid sequence (as depicted in FIG. 2J)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 73, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 74[LTG2322, LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM aminoacid sequence (as depicted in FIG. 2K)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 73 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 74 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2322, LP-mD1.22-CD8 TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM aminoacid sequence (as depicted in FIG. 2K)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 77, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 78[LTG2314, LP-mD1.22-L3-C46-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2L)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 77 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 78 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2314, LP-mD1.22-L3-C46-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2L)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 81, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 82[LTG2315, LP-mD1.22-L5-C46-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2M)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 81 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 82 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2315, LP-mD1.22-L5-C46-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2M)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 85, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 86[LTG2316, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2N)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 85 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 86 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2316, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2N)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 89, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 90[LTG2317, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2O)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 89 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 90 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2317, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta amino acid sequence (asdepicted in FIG. 2O)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 93, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 94[LTG2318, LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3zeta amino acidsequence (as depicted in FIG. 2P)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 93 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 94 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2318, LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3zeta amino acidsequence (as depicted in FIG. 2P)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 97, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 98[LTG2319, LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3zeta amino acidsequence (as depicted in FIG. 2Q)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 97 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 98 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2319, LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3zeta amino acidsequence (as depicted in FIG. 2Q)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 101, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 102[LTG2320, LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta amino acidsequence (as depicted in FIG. 2R)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 101 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 102 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2320, LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta amino acidsequence (as depicted in FIG. 2R)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 105, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 106[LTG2323, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TMamino acid sequence (as depicted in FIG. 2S)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 105 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 106 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2323, LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-C46-TNFRSF19TMamino acid sequence (as depicted in FIG. 2S)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 107, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 108[LTG2329,LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2amino acid sequence (as depicted in FIG. 2T)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 107 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 108 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2329,LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2amino acid sequence (as depicted in FIG. 2T)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 109, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 110[LTG2330,LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2amino acid sequence (as depicted in FIG. 2U)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 109 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 110 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2330,LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2amino acid sequence (as depicted in FIG. 2U)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 113, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 114[LTG2331, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TMamino acid sequence (as depicted in FIG. 2V)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 113 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 114 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2331, LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TMamino acid sequence (as depicted in FIG. 2V)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 117, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 118[LTG2332, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TMamino acid sequence (as depicted in FIG. 2W)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 117 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 118 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2332, LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TMamino acid sequence (as depicted in FIG. 2W)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 121, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 122[LTG2334, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-LP2-C46-TNFRSF19TMamino acid sequence (as depicted in FIG. 2X)].

In yet another embodiment, the nucleic acid sequence encoding a CARcomprises the nucleic acid sequence of SEQ ID NO: 121 or a sequence with85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes theCAR comprising the amino acid sequence as set forth in SEQ ID NO: 122 ora sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof[LTG2334, LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-LP2-C46-TNFRSF19TMamino acid sequence (as depicted in FIG. 2X)].

Overall, anti-HIV CARs were highly expressed on the surface of primaryT-cells (FIGS. 3A-B, 4, 11A-B, 12A, and 12C). The mD1.22-based CARs wereexceptionally potent and specifically destroyed 293T cells stablyexpressing HIV gp120 (herein referred to as, env⁺ cells), which servesas a surrogate HIV-positive cell line to quantify CAR-mediatedcytotoxicity (FIGS. 3C, 5, 8, 10, 11C, and 12B). To understand thecytotoxic contribution of each anti-HIV targeting moiety, anti-HIV CARswere engineered containing either the mD1.22 domain, m36.4 domain, orC46 peptide. These anti-HIV CARs differentially killed env cells (FIG.3C, mD1.22>m36.4>C46, P<0.0001) with no off-target cytotoxic effect(FIG. 3D) and were triggered to produce IFN-γ in the presence of env⁺and to some extent in its absence (FIG. 3E).

To define the most optimal bispecific architecture, the mD1.22 domainand the m36.4 domain were fused using a series of flexibleglycine-serine linkers or placed each binder on its own CD3 signalingdomain. As shown in FIG. 4, all linker-specific CARs as well as otherdesign iterations were detected on the surface of primary T-cells.Interestingly, a bispecific CAR significantly enhanced anti-HIVcytotoxic effect (FIG. 5, LTG2303 versus LTG1944 or LTG1945) whilemaintaining exceptional specificity (FIG. 6). Decreasing the linkerlength between the two domains increased CAR-mediated cytotoxicity ofbispecific CARs engineered with a single CD3 while maintainingspecificity (FIG. 8). However, a modest increase in tonic signaling wasobserved in the absence of env⁺ target cell as measured by cytokinerelease assay (FIG. 9). More importantly, no off-target killing wasobserved in the presence of Raji cells which are known to contain MHCclass II molecules that may potentially interact with the CD4-receptorderived mD1.22 domain (FIG. 6, bottom panel). Moreover, bispecificanti-HIV CARs released IFN-γ upon encountering HIV-envelope target cellsand to a lesser extent in its absence (FIG. 7, 9). Taken together, abispecific CAR serves as a potent architecture to engineer a moreadvanced trispecific CAR.

To build a trispecific CAR composed of mD1.22, m36.4 and the C46peptide, additional bispecific CAR iterations were developed, in, thoseengineered with the mD1.22 and C46 peptide. In contrast to thebispecific CARs, replacing m36.4 with C46 severely abrogated CARfunction when the C46 peptide was positioned proximal to the T-cellmembrane. Only when the C46 domain was positioned distal to the mD1.22domain, was anti-HIV CAR-mediated cytotoxicity restored (FIG. 10A).Reminiscent of the CD22 CAR previously described, binder accessibilityrelative to the distance from the T-cell membrane is essential for CARfunction (Haso et al., Blood 2013; 121:1165-1174). As expected,abrogated CAR function as a result of mD1.22 and C46 binderconfiguration also led to poor levels of IFN-γ secretion (FIG. 10B).Strikingly, merely reversing the order of the domains restored highlevel IFN-γ secretion (LTG2316) while extending the space between thetwo domains led to lower tonic signaling (LTG2317). Taken together,these data clearly reveal the most optimal bispecific binderconfiguration for a C46-based bispecific CAR. More importantly, a set ofrules that govern bispecific CAR function were identified that can beapplied to rationally design a trispecific anti-HIV CAR.

Targeting multiple epitope determinants on the HIV envelope protein isan attractive strategy to engineer CARs with superior breadth, potency,and ability to prevent emergence of escape mutants. The rationale fordeveloping a trispecific CAR is that if one binder is unable torecognize a HIV variant then the second and/or third domain wouldcompensate for this loss of function acquired by the CAR. Thus, toimprove the breadth of the most potent bispecific CAR candidates, thebispecific CAR was engineered with a third highly potent fusioninhibitor (C46 peptide) or entry inhibitor (m36.4) to form a trispecificCAR. As compared to bispecific CAR, trispecific CARs maintained theirability to potently destroy the HIV surrogate cell line and initiated arobust Th1 cytokine response (FIGS. 11C and 11E-F). Moreover,trispecific CARs exhibited exceptional specificity for their intendedtargets with no off-target effect on Raji cells (FIG. 11D). As shownfrom the data, the most optimal trispecific CAR was LTG2323 followed byLTG2320. Further evaluation of two additional trispecific CARs generatedby combining the LTG2303 and LTG2316 or LTG2317 architectures againdemonstrated robust killing of the surrogate HIV envelope cell line(FIG. 12B). Except for the highest E:T ratio, both LTG2329 and LTG2330maintained their specificity (FIG. 12D). Taken together, the precisearchitecture of the trispecific CAR improved its anti-HIV function.

Subsequent challenge of anti-HIV CART cells with diverse and resistantHIV-1 strains further confirmed the importance of the anti-HIV CARarchitecture. Starting with a CD4⁺ enriched T cell population frommultiple HIV-naïve donors (FIG. 13), CAR T cell products were generatedfor a select group of anti-HIV CARs. Using a modified in vitro HIV-1Env-IMC-LucR challenge assay, the most potent anti-HIV CARs wereidentified as bispecific and trispecific CARs containing two CD3 chains(hereafter referred to as duoCAR). As shown in FIGS. 14-17, bispecificand trispecific duoCARs were superior to conventional anti-HIV CARscontaining a single CD3 chain irrespective of its valency (LTG2303,LTG2329, and LTG2330). This is an important design feature fordeveloping anti-HIV CARs to evade viral escape while increasing potencyand breadth. It is presumed that if one domain is lost due to mutationalescape, the other domain may compensate for this loss. More importantly,the anti-HIV targeting domains(s) may act independent of each other,sequentially, or simultaneously by virtue of the duoCAR architecture toattack the productive HIV-infected cell. As shown in FIG. 18, primary Tcells engineered with the mD1.22-CAR were more susceptible to HIV-1infection than bispecific or trispecific CAR-T cells (open red bar). Theincorporation of the m36.4 domain, which is an entry inhibitor, wassufficient to protect CAR T cells and ablate HIV-1 infection. To furtherevaluate bispecific and trispecific duoCAR-T cells in vivo, a humanizedNSG mouse model (hu-spl-PBMC-NSG) of acute and chronic HIV-1 infectionwas employed (FIG. 19A). The VRC01/3BNC117-resistant Env-IMC-LucR viruswas selected to further interrogate duoCAR-T cell function. As shown inFIGS. 19B, C, bispecific and trispecific duoCARs significantly reducedHIV-1 infection as compared to the UTD-treated, HIV-infected cohort.Both bispecific and trispecific duoCARs demonstrated similar potencies(LTG2303 versus LTG2330). In the chronic HIV infection study, the CD4⁺ Tcells were significantly depleted in the spleens of mice treated withthe control UTD T cells (FIG. 19E). Conversely, mice treated with thebispecific and trispecific duoCAR-T cells showed significant improvementin the % of CD4⁺ T cells recovered from infected spleens whichapproached levels greater than or similar to the uninfected mice (seeHIV-PBMC, FIG. 19E). Concomitant with CD4⁺ T cell depletion duringchronic HIV infection was an increase in % of CD8⁺ T cells within thespleens of infected, UTD-treated mice with uncontrolled HIV infection(FIG. 19G). The robust control of HIV-1 infection is likely due to thein vivo persistence of the CAR-T cells within the spleens of infectedmice as shown in FIGS. 19H, I. Collectively, the invention presentedherein represents a powerful and universal multi-targeting HIV-1immunotherapy with strong implication for a functional cure.

Without being intended to limit to any particular mechanism of action,it is believed that possible reasons for the enhanced therapeuticfunction associated with the exemplary CARs of the invention include,for example, and not by way of limitation, a) improved multi-specifictargeting of non-redundant viral epitopes, b) rationale CAR design usingintracellular T-cell signaling domains to leverage function of antigenbinding domains, c) lateral movement within the plasma membrane allowingfor more efficient signal transduction, d) superior location withinplasma membrane microdomains, such as lipid rafts, and greater abilityto interact with transmembrane signaling cascades associated with T cellactivation, e) superior location within the plasma membrane bypreferential movement away from dampening or down-modulatoryinteractions, such as less proximity to or interaction with phosphatasessuch as CD45, and f) superior assembly into T cell receptor signalingcomplexes (i.e. the immune synapse), or any combination thereof.

While the disclosure has been illustrated with precise combinations ofthree exemplary HIV envelope targeting domains (mD1.22, m36.4, and C46peptide), other nucleotide and/or amino acid variants within thesebinding domains may be used to derive the HIV envelope binding domainsfor use in the CARs described herein.

Depending on the desired antigen to be targeted, the CAR can beadditionally engineered to include the appropriate antigen bindingdomain that is specific to the desired antigen target.

In one aspect of the present invention, there is provided a CAR capableof binding to a non-TSA or non-TAA including, for example and not by wayof limitation, an antigen derived from Retroviridae (e.g. humanimmunodeficiency viruses such as HIV-1 and HIV-LP), Picornaviridae (e.g.poliovirus, hepatitis A virus, enterovirus, human coxsackievirus,rhinovirus, and echovirus), rubella virus, coronavirus, vesicularstomatitis virus, rabies virus, ebola virus, parainfluenza virus, mumpsvirus, measles virus, respiratory syncytial virus, influenza virus,hepatitis B virus, parvovirus, Adenoviridae, Herpesviridae [e.g. type 1and type 2 herpes simplex virus (HSV), varicella-zoster virus, EpsteinBarr virus (EBV), cytomegalovirus (CMV), and herpes virus], Poxviridae(e.g. smallpox virus, vaccinia virus, and pox virus), or hepatitis Cvirus, or any combination thereof.

In another aspect of the present invention, there is provided a CARcapable of binding to an antigen derived from a bacterial strain ofStaphylococci, Streptococcus, Escherichia coli, Pseudomonas, orSalmonella. Particularly, there is provided a CAR capable of binding toan antigen derived from an infectious bacterium, for example,Helicobacter pylori, Legionella pneumophilia, a bacterial strain ofMycobacteria species. (e.g. M. tuberculosis, M. avium, M.intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes,Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus(Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridiumtetani, or a combination thereof.

In another aspect of the present invention, there is provided a CAR incombination with advanced gene editing technologies (e.g., CRISPR/Cas9,CRISPR/Cas13, riboswitch, RNA interference, or intrabody) that excisesand/or interferes with viral ribonucleic acids, integrated viral DNA(for example, HIV provirus), and/or viral proteins (e.g., viral reversetranscriptase) from host-infected cells, or in combination thereof, withadvanced gene editing technologies to modulate host genetic factorsimplicated in the disease such as, chemokine receptor G-protein coupledreceptors (e.g., CXCR4, CCR5), host susceptibility factors (e.g.,LEDGF/p75), virulence factors (e.g., DC-SIGN), natural host resistancefactors (e.g., Defensins), or a combination thereof, but not limited tothe aforementioned combinations.

In another aspect of the present invention, there is provided a CAR incombination with a small molecule inhibitor, latency-reversing agent,anti-viral agent, anti-microbial agent, or antibody, and any derivativethereof, that potentiates and/or synergizes CAR-T function (e.g., TLR7agonist), targets a disease-state related to CAR therapy, or acombination thereof, but not limited to the aforementioned combinations.

2. Transmembrane Domain

With respect to the transmembrane domain, the CAR comprises one or moretransmembrane domains fused to the extracellular mD1.22 and m36.4antigen binding domain of the CAR.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein.

Transmembrane regions of particular use in the CARs described herein maybe derived from (i.e. comprise at least the transmembrane region(s) of)the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon,CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64,CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19.Alternatively, the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

In one embodiment, the transmembrane domain that naturally is associatedwith one of the domains in the CAR is used in addition to thetransmembrane domains described supra.

In some instances, the transmembrane domain can be selected or by aminoacid substitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex.

In one embodiment, the transmembrane domain in the CAR of the inventionis the CD8 transmembrane domain. In one embodiment, the CD8transmembrane domain comprises the nucleic acid sequence of SEQ ID NO:7. In one embodiment, the CD8 transmembrane domain comprises the nucleicacid sequence that encodes the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the encoded transmembrane domain comprises an aminoacid sequence having at least one, two or three modifications (e.g.,substitutions) but not more than 20, 10 or 5 modifications (e.g.,substitutions) of an amino acid sequence of SEQ ID NO: 8, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO: 8.

In some instances, the transmembrane domain of the CAR comprises theCD8.alpha.hinge domain. In one embodiment, the CD8 hinge domaincomprises the nucleic acid sequence of SEQ ID NO: 9. In one embodiment,the CD8 hinge domain comprises the nucleic acid sequence that encodesthe amino acid sequence of SEQ ID NO: 10. In another embodiment, the CD8hinge domain comprises the amino acid sequence of SEQ ID NO: 10, or asequence with 95-99% identify thereof.

In one embodiment, an isolated nucleic acid molecule is provided whereinthe encoded linker domain is derived from the extracellular domain ofCD8, and is linked to the transmembrane CD8 domain, the transmembraneTNFRSF19 domain, or a combination thereof.

In one embodiment, the encoded transmembrane TNFRSF19 domain incombination with the CD8 linker/hinge domain comprises an amino acidsequence having at least one, two or three modifications (e.g.,substitutions) but not more than 20, 10 or 5 modifications (e.g.,substitutions) of an amino acid sequence of SEQ ID NO: 16, or a sequencewith 95-99% identity to an amino acid sequence of SEQ ID NO: 16.

3. Spacer Domain

In the CAR, a spacer domain can be arranged between the extracellulardomain and the transmembrane domain, or between the intracellular domainand the transmembrane domain. The spacer domain means any oligopeptideor polypeptide that serves to link the transmembrane domain with theextracellular domain and/or the transmembrane domain with theintracellular domain. The spacer domain comprises up to 300 amino acids,preferably 10 to 100 amino acids, and most preferably 25 to 50 aminoacids.

In several embodiments, the linker can include a spacer element, which,when present, increases the size of the linker such that the distancebetween the effector molecule or the detectable marker and the antibodyor antigen binding fragment is increased. Exemplary spacers are known tothe person of ordinary skill, and include those listed in U.S. Pat. Nos.7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065,5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725,5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973,4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well asU.S. Pat. Pub. Nos. 20110212088 and 20110070248, each of which isincorporated by reference herein in its entirety.

The spacer domain preferably has a sequence that promotes binding of aCAR with an antigen and enhances signaling into a cell. Examples of anamino acid that is expected to promote the binding include cysteine, acharged amino acid, and serine and threonine in a potentialglycosylation site, and these amino acids can be used as an amino acidconstituting the spacer domain.

As the spacer domain, the entire or a part of amino acid numbers 137-206(SEQ ID NO: 10) which is a hinge region of CD8.alpha. (NCBI RefSeq:NP.sub.--001759.3), amino acid numbers 135 to 195 of CD8.beta. (GenBank:AAA35664.1), amino acid numbers 315 to 396 of CD4 (NCBI RefSeq:NP.sub.--000607.1), or amino acid numbers 137 to 152 of CD28 (NCBIRefSeq: NP.sub.--006130.1) can be used. Also, as the spacer domain, apart of a constant region of an antibody H chain or L chain can be used.Further, the spacer domain may be an artificially synthesized sequence.

Further, in the CAR, a signal peptide sequence can be linked to theN-terminus. The signal peptide sequence exists at the N-terminus of manysecretory proteins and membrane proteins, and has a length of 15 to 30amino acids. Since many of the protein molecules mentioned above as theintracellular domain have signal peptide sequences, the signal peptidescan be used as a signal peptide for the CAR. In one embodiment, thesignal peptide comprises the amino acid sequence shown in SEQ ID NO: 36.In another embodiment, the signal peptide comprises the amino acidsequence in SEQ ID NO: 38.

4. Intracellular Domain

The cytoplasmic domain or otherwise the intracellular signaling domainof the CAR is responsible for activation of at least one of the normaleffector functions of the immune cell in which the CAR has been placedin. The term “effector function” refers to a specialized function of acell. Effector function of a T cell, for example, may be cytolyticactivity or helper activity including the secretion of cytokines. Thusthe term “intracellular signaling domain” refers to the portion of aprotein which transduces the effector function signal and directs thecell to perform a specialized function. While usually the entireintracellular signaling domain can be employed, in many cases it is notnecessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The term intracellularsignaling domain is thus meant to include any truncated portion of theintracellular signaling domain sufficient to transduce the effectorfunction signal.

Preferred examples of intracellular signaling domains for use in the CARinclude the cytoplasmic sequences of the T cell receptor (TCR) andco-receptors that act in concert to initiate signal transductionfollowing antigen receptor engagement, as well as any derivative orvariant of these sequences and any synthetic sequence that has the samefunctional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

Examples of ITAM containing primary cytoplasmic signaling sequences thatare of particular use in the CARs disclosed herein include those derivedfrom TCR zeta (CD3 Zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific, non-limitingexamples, of the ITAM include peptides having sequences of amino acidnumbers 51 to 164 of CD3.zeta. (NCBI RefSeq: NP.sub.--932170.1), aminoacid numbers 45 to 86 of Fc.epsilon.RI.gamma. (NCBI RefSeq:NP.sub.--004097.1), amino acid numbers 201 to 244 of Fc.epsilon.RI.beta.(NCBI RefSeq: NP.sub.--000130.1), amino acid numbers 139 to 182 ofCD3.gamma. (NCBI RefSeq: NP.sub.--000064.1), amino acid numbers 128 to171 of CD3.delta. (NCBI RefSeq: NP.sub.--000723.1), amino acid numbers153 to 207 of CD3.epsilon. (NCBI RefSeq: NP.sub.--000724.1), amino acidnumbers 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acidnumbers 707 to 847 of 0022 (NCBI RefSeq: NP.sub.--001762.2), amino acidnumbers 166 to 226 of CD79a (NCBI RefSeq: NP.sub.--001774.1), amino acidnumbers 182 to 229 of CD79b (NCBI RefSeq: NP.sub.--000617.1), and aminoacid numbers 177 to 252 of CD66d (NCBI RefSeq: NP.sub.--001806.2), andtheir variants having the same function as these peptides have. Theamino acid number based on amino acid sequence information of NCBIRefSeq ID or GenBank described herein is numbered based on the fulllength of the precursor (comprising a signal peptide sequence etc.) ofeach protein. In one embodiment, the cytoplasmic signaling molecule inthe CAR comprises a cytoplasmic signaling sequence derived from CD3zeta.

In a preferred embodiment, the intracellular domain of the CAR can bedesigned to comprise the CD3-zeta signaling domain by itself or combinedwith any other desired cytoplasmic domain(s) useful in the context ofthe CAR. For example, the intracellular domain of the CAR can comprise aCD3 zeta chain portion and a costimulatory signaling region. Thecostimulatory signaling region refers to a portion of the CAR comprisingthe intracellular domain of a costimulatory molecule. A costimulatorymolecule is a cell surface molecule other than an antigen receptor ortheir ligands that is required for an efficient response of lymphocytesto an antigen. Examples of such costimulatory molecules include CD27,CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and a ligand that specifically binds with CD83, and the like. Specific,non-limiting examples, of such costimulatory molecules include peptideshaving sequences of amino acid numbers 236 to 351 of CD2 (NCBI RefSeq:NP.sub.--001758.2), amino acid numbers 421 to 458 of CD4 (NCBI RefSeq:NP.sub.--000607.1), amino acid numbers 402 to 495 of CD5 (NCBI RefSeq:NP.sub.--055022.2), amino acid numbers 207 to 235 of CD8.alpha. (NCBIRefSeq: NP.sub.--001759.3), amino acid numbers 196 to 210 of CD83(GenBank: AAA35664.1), amino acid numbers 181 to 220 of CD28 (NCBIRefSeq: NP.sub.--006130.1), amino acid numbers 214 to 255 of CD137(4-1BB, NCBI RefSeq: NP.sub.--001552.2), amino acid numbers 241 to 277of CD134 (OX40, NCBI RefSeq: NP.sub.--003318.1), and amino acid numbers166 to 199 of ICOS (NCBI RefSeq: NP.sub.--036224.1), and their variantshaving the same function as these peptides have. Thus, while thedisclosure herein is exemplified primarily with 4-1BB as theco-stimulatory signaling element, other costimulatory elements arewithin the scope of the disclosure.

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR may be linked to each other in a random or specifiedorder. Optionally, a short oligo- or polypeptide linker, preferablybetween 2 and 10 amino acids in length may form the linkage. Aglycine-serine doublet provides a particularly suitable linker.

In one embodiment, the intracellular domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28. Inanother embodiment, the intracellular domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of 4-1BB. In yetanother embodiment, the intracellular domain is designed to comprise thesignaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.

In one embodiment, the intracellular domain in the CAR is designed tocomprise the signaling domain of 4-1BB and the signaling domain ofCD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleicacid sequence set forth in SEQ ID NO: 17 and the signaling domain ofCD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 19.

In one embodiment, the intracellular domain in the CAR is designed tocomprise the signaling domain of 4-1BB and the signaling domain ofCD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleicacid sequence that encodes the amino acid sequence of SEQ ID NO: 18 andthe signaling domain of CD3-zeta comprises the nucleic acid sequencethat encodes the amino acid sequence of SEQ ID NO: 20.

In one embodiment, the intracellular domain in the CAR is designed tocomprise the codon degenerate signaling domain of CD3-zeta2, wherein thesignaling domain of CD3-zeta2 comprises the nucleic acid sequence SEQ IDNO: 21 and the amino acid sequence set forth in SEQ ID NO: 22.

5. Additional Description of CARs

Also expressly included within the scope of the invention are functionalportions of the CARs disclosed herein. The term “functional portion”when used in reference to a CAR refers to any part or fragment of one ormore of the CARs disclosed herein, which part or fragment retains thebiological activity of the CAR of which it is a part (the parent CAR).Functional portions encompass, for example, those parts of a CAR thatretain the ability to recognize target cells, or detect, treat, orprevent a disease, to a similar extent, the same extent, or to a higherextent, as the parent CAR. In reference to the parent CAR, thefunctional portion can comprise, for instance, about 10%, 25%, 30%, 50%,68%, 80%, 90%, 95%, or more, of the parent CAR.

The functional portion can comprise additional amino acids at the aminoor carboxyl terminus of the portion, or at both termini, whichadditional amino acids are not found in the amino acid sequence of theparent CAR. Desirably, the additional amino acids do not interfere withthe biological function of the functional portion, e.g., recognizetarget cells, detect cancer, treat or prevent cancer, etc. Moredesirably, the additional amino acids enhance the biological activity,as compared to the biological activity of the parent CAR.

Included in the scope of the disclosure are functional variants of theCARs disclosed herein. The term “functional variant” as used hereinrefers to a CAR, polypeptide, or protein having substantial orsignificant sequence identity or similarity to a parent CAR, whichfunctional variant retains the biological activity of the CAR of whichit is a variant. Functional variants encompass, for example, thosevariants of the CAR described herein (the parent CAR) that retain theability to recognize target cells to a similar extent, the same extent,or to a higher extent, as the parent CAR. In reference to the parentCAR, the functional variant can, for instance, be at least about 30%,50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to theparent CAR.

A functional variant can, for example, comprise the amino acid sequenceof the parent CAR with at least one conservative amino acidsubstitution. Alternatively or additionally, the functional variants cancomprise the amino acid sequence of the parent CAR with at least onenon-conservative amino acid substitution. In this case, it is preferablefor the non-conservative amino acid substitution to not interfere withor inhibit the biological activity of the functional variant. Thenon-conservative amino acid substitution may enhance the biologicalactivity of the functional variant, such that the biological activity ofthe functional variant is increased as compared to the parent CAR.

Amino acid substitutions of the CARs are preferably conservative aminoacid substitutions. Conservative amino acid substitutions are known inthe art, and include amino acid substitutions in which one amino acidhaving certain physical and/or chemical properties is exchanged foranother amino acid that has the same or similar chemical or physicalproperties. For instance, the conservative amino acid substitution canbe an acidic/negatively charged polar amino acid substituted for anotheracidic/negatively charged polar amino acid (e.g., Asp or Glu), an aminoacid with a nonpolar side chain substituted for another amino acid witha nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp,Cys, Val, etc.), a basic/positively charged polar amino acid substitutedfor another basic/positively charged polar amino acid (e.g. Lys, His,Arg, etc.), an uncharged amino acid with a polar side chain substitutedfor another uncharged amino acid with a polar side chain (e.g., Asn,Gin, Ser, Thr, Tyr, etc.), an amino acid with a beta-branched side-chainsubstituted for another amino acid with a beta-branched side-chain(e.g., He, Thr, and Val), an amino acid with an aromatic side-chainsubstituted for another amino acid with an aromatic side chain (e.g.,His, Phe, Trp, and Tyr), etc.

The CAR can consist essentially of the specified amino acid sequence orsequences described herein, such that other components, e.g., otheramino acids, do not materially change the biological activity of thefunctional variant.

The CARs (including functional portions and functional variants) can beof any length, i.e., can comprise any number of amino acids, providedthat the CARs (or functional portions or functional variants thereof)retain their biological activity, e.g., the ability to specifically bindto antigen, detect diseased cells in a mammal, or treat or preventdisease in a mammal, etc. For example, the CAR can be about 50 to about5000 amino acids long, such as 50, 70, 75, 100, 125, 150, 175, 200, 300,400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

The CARs (including functional portions and functional variants of theinvention) can comprise synthetic amino acids in place of one or morenaturally-occurring amino acids. Such synthetic amino acids are known inthe art, and include, for example, aminocyclohexane carboxylic acid,norleucine, -amino n-decanoic acid, homoserine,S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline,4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine,4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine,phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine,indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, aminomalonic acid, aminomalonic acid monoamide,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptanecarboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid,γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, anda-tert-butylglycine.

The CARs (including functional portions and functional variants) can beglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated, cyclized via, e.g., a disulfide bridge, or converted into anacid addition salt and/or optionally dimerized or polymerized, orconjugated.

The CARs (including functional portions and functional variants thereof)can be obtained by methods known in the art. The CARs may be made by anysuitable method of making polypeptides or proteins. Suitable methods ofde novo synthesizing polypeptides and proteins are described inreferences, such as Chan et al., Fmoc Solid Phase Peptide Synthesis,Oxford University Press, Oxford, United Kingdom, 2000; Peptide andProtein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; EpitopeMapping, ed. Westwood et al., Oxford University Press, Oxford, UnitedKingdom, 2001; and U.S. Pat. No. 5,449,752. Also, polypeptides andproteins can be recombinantly produced using the nucleic acids describedherein using standard recombinant methods. See, for instance, Sambrooket al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold SpringHarbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, N Y, 1994. Further, some of the CARs (including functionalportions and functional variants thereof) can be isolated and/orpurified from a source, such as a plant, a bacterium, an insect, amammal, e.g., a rat, a human, etc. Methods of isolation and purificationare well-known in the art. Alternatively, the CARs described herein(including functional portions and functional variants thereof) can becommercially synthesized by companies. In this respect, the CARs can besynthetic, recombinant, isolated, and/or purified.

B. Antibodies and Antigen Binding Fragments

One embodiment further provides a CAR, a T cell expressing a CAR, anantibody, or antigen binding domain or portion thereof, whichspecifically binds to one or more of the antigens disclosed herein. Asused herein, a “T cell expressing a CAR,” or a “CAR T cell” or a “CAR-T”means a T cell expressing a CAR, and has antigen specificity determinedby, for example, the antibody-derived targeting domain of the CAR.

As used herein, and “antigen binding domain” can include an antibody andantigen binding fragments thereof. The term “antibody” is used herein inthe broadest sense and encompasses various antibody structures,including but not limited to monoclonal antibodies, polyclonalantibodies, multi-specific antibodies (e.g., bispecific antibodies), andantigen binding fragments thereof, so long as they exhibit the desiredantigen-binding activity. Non-limiting examples of antibodies include,for example, intact immunoglobulins and variants and fragments thereofknown in the art that retain binding affinity for the antigen.

A “monoclonal antibody” is an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic epitope. The modifier “monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. In some examples, amonoclonal antibody is an antibody produced by a single clone of Blymphocytes or by a cell into which nucleic acid encoding the light andheavy variable regions of the antibody of a single antibody (or anantigen binding fragment thereof) have been transfected, or a progenythereof. In some examples monoclonal antibodies are isolated from asubject. Monoclonal antibodies can have conservative amino acidsubstitutions which have substantially no effect on antigen binding orother immunoglobulin functions. Exemplary methods of production ofmonoclonal antibodies are known, for example, see Harlow & Lane,Antibodies, A Laboratory Manual, 2nd ed. Cold Spring HarborPublications, New York (2013).

Typically, an immunoglobulin has heavy (H) chains and light (L) chainsinterconnected by disulfide bonds. Immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as the myriad immunoglobulin variable domain genes. Thereare two types of light chain, lambda (λ) and kappa (κ). There are fivemain heavy chain classes (or isotypes) which determine the functionalactivity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region (or constantdomain) and a variable region (or variable domain; see, e.g., Kindt etal. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91 (2007).)In several embodiments, the heavy and the light chain variable regionscombine to specifically bind the antigen. In additional embodiments,only the heavy chain variable region is required. For example, naturallyoccurring camelid antibodies consisting of a heavy chain only arefunctional and stable in the absence of light chain (see, e.g.,Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat.Struct. Biol., 3:733-736, 1996). References to “VH” or “VH” refer to thevariable region of an antibody heavy chain, including that of an antigenbinding fragment, such as Fv, ScFv, dsFv or Fab. References to “VL” or“VL” refer to the variable domain of an antibody light chain, includingthat of an Fv, ScFv, dsFv or Fab.

Light and heavy chain variable regions contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs” (see, e.g., Kabat etal., Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. The framework region of an antibody, that is the combinedframework regions of the constituent light and heavy chains, serves toposition and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The amino acid sequence boundaries of a given CDR can bereadily determined using any of a number of well-known schemes,including those described by Kabat et al. (“Sequences of Proteins ofImmunological Interest,” 5^(th) Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991; “Kabat” numbering scheme),Al-Lazikani et al., (JMB 273,927-948, 1997; “Chothia” numbering scheme),and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains,” Dev. Comp.Immunol., 27:55-77, 2003; “IMGT” numbering scheme). The CDRs of eachchain are typically referred to as CDR1, CDR2, and CDR3 (from theN-terminus to C-terminus), and are also typically identified by thechain in which the particular CDR is located. Thus, a VH CDR3 is theCDR3 from the variable domain of the heavy chain of the antibody inwhich it is found, whereas a VL CDR1 is the CDR1 from the variabledomain of the light chain of the antibody in which it is found. Lightchain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavychain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

An “antigen binding fragment” is a portion of a full length antibody orportion of a full length protein (for example, the D1 domain of the CD4receptor) that retains the ability to specifically recognize the cognateantigen, as well as various combinations of such portions. Non-limitingexamples of antigen binding fragments include protein domains,full-length proteins, Fv, Fab, Fab′, Fab′-SH, F(ab′)2; diabodies;nanobodies; linear antibodies; single-chain antibody molecules (e.g.ScFv); and multi-specific antibodies formed from antibody fragments ormulti-specific proteins formed from more than one protein domain orfragment. Antibody fragments include antigen binding fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies (see, e.g., Kontermann andDubel (Ed), Antibody Engineering, Vols. 1-2, 2nd Ed., Springer Press,2010). Multi-specific proteins and derivatives thereof include antigenbinding fragments produced by fusing the fragments together in a preciseconfiguration using the original fragments, modifications of thosefragments, or those synthesized de novo using recombinant DNAtechnologies.

A single-chain antibody (ScFv) is a genetically engineered moleculecontaining the VH and VL domains of one or more antibody(ies) linked bya suitable polypeptide linker as a genetically fused single chainmolecule (see, for example, Bird et al., Science, 242:423 426, 1988;Huston et al., Proc. Natl. Acad. Sci., 85:5879 5883, 1988; Ahmad et al.,Clin. Dev. Immunol., 2012, doi:10.1155/2012/980250; Marbry, IDrugs,13:543-549, 2010). The intramolecular orientation of the VH-domain andthe VL-domain in a ScFv, is typically not decisive for ScFvs. Thus,ScFvs with both possible arrangements (VH-domain-linkerdomain-VL-domain; VL-domain-linker domain-VH-domain) may be used.

In a dsFv, the heavy and light chain variable chains have been mutatedto introduce a disulfide bond to stabilize the association of thechains. Diabodies also are included, which are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see, for example, Holliger et al., Proc.Natl. Acad. Sci., 90:6444 6448, 1993; Poljak et al., Structure, 2:11211123, 1994).

Antibodies also include genetically engineered forms such as chimericantibodies (such as humanized murine antibodies) and heteroconjugateantibodies (such as bispecific antibodies). See also, Pierce Catalog andHandbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

Non-naturally occurring antibodies or antigen binding fragments can beconstructed using solid phase peptide synthesis, can be producedrecombinantly, or can be obtained, for example, by screeningcombinatorial libraries consisting of variable heavy chains and variablelight chains as described by Huse et al., Science 246:1275-1281 (1989),which is incorporated herein by reference. These and other methods ofmaking, for example, chimeric, humanized, CDR-grafted, single chain, andbifunctional antibodies or multifunctional binding domains, are wellknown to those skilled in the art (Winter and Harris, Immunol. Today14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow andLane, supra, 1988; Hilyard et al., Protein Engineering: A practicalapproach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed.(Oxford University Press 1995); each of which is incorporated herein byreference).

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. Antibody competition assays are known,and an exemplary competition assay is provided herein.

A “humanized” antibody or antigen binding fragment includes a humanframework region and one or more CDRs from a non-human (such as a mouse,rat, or synthetic) antibody or antigen binding fragment. The non-humanantibody or antigen binding fragment providing the CDRs is termed a“donor,” and the human antibody or antigen binding fragment providingthe framework is termed an “acceptor.” In one embodiment, all the CDRsare from the donor immunoglobulin in a humanized immunoglobulin.Constant regions need not be present, but if they are, they can besubstantially identical to human immunoglobulin constant regions, suchas at least about 85-90%, such as about 95% or more identical. Hence,all parts of a humanized antibody or antigen binding fragment, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human antibody sequences.

A “chimeric antibody” is an antibody which includes sequences derivedfrom two different antibodies, which typically are of different species.In some examples, a chimeric antibody includes one or more CDRs and/orframework regions from one human antibody and CDRs and/or frameworkregions from another human antibody.

A “fully human antibody” or “human antibody” or humanized derivativethereof consisting of a protein fragment is an antibody or derivativethereof which includes sequences from (or derived from) the humangenome, and does not include sequence from another species. In someembodiments, a human antibody includes CDRs, framework regions, and (ifpresent) an Fc region from (or derived from) the human genome. Humanantibodies can be identified and isolated using technologies forcreating antibodies based on sequences derived from the human genome,for example by phage display or using transgenic animals (see, e.g.,Barbas et al. Phage display: A Laboratory Manuel. 1st Ed. New York: ColdSpring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech.,23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a bispecific or bifunctional antibody has twodifferent binding sites while a trispecific or trifunctional antibodyhas three different binding sites.

Methods of testing antibodies for the ability to bind to any functionalportion of the CAR are known in the art and include any antibody-antigenbinding assay, such as, for example, radioimmunoassay (RIA), ELISA,Western blot, immunoprecipitation, and competitive inhibition assays(see, e.g., Janeway et al., infra, U.S. Patent Application PublicationNo. 2002/0197266 Al, and U.S. Pat. No. 7,338,929).

Also, a CAR, a T cell expressing a CAR, an antibody, or antigen bindingportion thereof, can be modified to comprise a detectable label, suchas, for instance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

C. Conjugates

A CAR, a T cell expressing a CAR, or monoclonal antibodies, or antigenbinding fragments thereof, specific for one or more of the antigensdisclosed herein, can be conjugated to an agent, such as an effectormolecule or detectable marker, using any number of means known to thoseof skill in the art. Both covalent and noncovalent attachment means maybe used. Conjugates include, but are not limited to, molecules in whichthere is a covalent linkage of an effector molecule or a detectablemarker to an antibody or antigen binding fragment that specificallybinds one or more of the antigens disclosed herein. One of skill in theart will appreciate that various effector molecules and detectablemarkers can be used, including (but not limited to) anti-viral agents,anti-microbial agents, chemotherapeutic agents, anti-angiogenic agents,toxins, radioactive agents such as ¹²⁵I, ³²P, ¹⁴C, ³H and ³⁵S and otherlabels, target moieties and ligands, etc.

The choice of a particular effector molecule or detectable markerdepends on the particular target molecule or cell, and the desiredbiological effect. Thus, for example, the effector molecule can be acytotoxin that is used to bring about the death of a particular targetcell (such as a viral-infected cell).

The procedure for attaching an effector molecule or detectable marker toan antibody or antigen binding fragment varies according to the chemicalstructure of the effector. Polypeptides typically contain a variety offunctional groups; such as carboxylic acid (COOH), free amine (−NH₂) orsulfhydryl (—SH) groups, which are available for reaction with asuitable functional group on an antibody to result in the binding of theeffector molecule or detectable marker. Alternatively, the antibody orantigen binding fragment is a derivative to expose or attach additionalreactive functional groups. The derivatization may involve attachment ofany of a number of known linker molecules such as those available fromPierce Chemical Company, Rockford, Ill. The linker can be any moleculeused to join the antibody or antigen binding fragment to the effectormolecule or detectable marker. The linker is capable of forming covalentbonds to both the antibody or antigen binding fragment and to theeffector molecule or detectable marker. Suitable linkers are well knownto those of skill in the art and include, but are not limited to,straight or branched-chain carbon linkers, heterocyclic carbon linkers,or peptide linkers. Where the antibody or antigen binding fragment andthe effector molecule or detectable marker are polypeptides, the linkersmay be joined to the constituent amino acids through their side groups(such as through a disulfide linkage to cysteine) or to the alpha carbonamino and carboxyl groups of the terminal amino acids.

In several embodiments, the linker can include a spacer element, which,when present, increases the size of the linker such that the distancebetween the effector molecule or the detectable marker and the antibodyor antigen binding fragment is increased. Exemplary spacers are known tothe person of ordinary skill, and include those listed in U.S. Pat. Nos.7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065,5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725,5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973,4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well asU.S. Pat. Pub. Nos. 20110212088 and 20110070248, each of which isincorporated by reference herein in its entirety.

In some embodiments, the linker is cleavable under intracellularconditions, such that cleavage of the linker releases the effectormolecule or detectable marker from the antibody or antigen bindingfragment in the intracellular environment. In yet other embodiments, thelinker is not cleavable and the effector molecule or detectable markeris released, for example, by antibody degradation. In some embodiments,the linker is cleavable by a cleaving agent that is present in theintracellular environment (for example, within a lysosome or endosome orcaveolea). The linker can be, for example, a peptide linker that iscleaved by an intracellular peptidase or protease enzyme, including, butnot limited to, a lysosomal or endosomal protease. In some embodiments,the peptide linker is at least two amino acids long or at least threeamino acids long. However, the linker can be 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or 15 amino acids long, such as 1-2, 1-3, 2-5, 3-10, 3-15,1-5, 1-10, 1-15 amino acids long. Proteases can include cathepsins B andD and plasmin, all of which are known to hydrolyze dipeptide drugderivatives resulting in the release of active drug inside target cells(see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics83:67-123). For example, a peptide linker that is cleavable by thethiol-dependent protease cathepsin-B, can be used (for example, aPhenylalanine-Leucine or a Glycine-Phenylalanine-Leucine-Glycinelinker). Other examples of such linkers are described, for example, inU.S. Pat. No. 6,214,345, incorporated herein by reference. In a specificembodiment, the peptide linker cleavable by an intracellular protease isa Valine-Citruline linker or a Phenylalanine-Lysine linker (see, forexample, U.S. Pat. No. 6,214,345, which describes the synthesis ofdoxorubicin with the Valine-Citruline linker).

In other embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker is hydrolysable under acidic conditions. Forexample, an acid-labile linker that is hydrolysable in the lysosome (forexample, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconiticamide, orthoester, acetal, ketal, or the like) can be used. (See, forexample, U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik andWalker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol.Chem. 264:14653-14661.) Such linkers are relatively stable under neutralpH conditions, such as those in the blood, but are unstable at below pH5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments,the hydrolysable linker is a thioether linker (such as, for example, athioether attached to the therapeutic agent via an acylhydrazone bond(see, for example, U.S. Pat. No. 5,622,929).

In other embodiments, the linker is cleavable under reducing conditions(for example, a disulfide linker). A variety of disulfide linkers areknown in the art, including, for example, those that can be formed usingSATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-,SPDB and SMPT. (See, for example, Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:92809290, 2008).See also U.S. Pat. No. 4,880,935.)

In yet other specific embodiments, the linker is a malonate linker(Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyllinker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a3′-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker is not cleavable and the effectormolecule or detectable marker is released by antibody degradation. (SeeU.S. Publication No. 2005/0238649 incorporated by reference herein inits entirety).

In several embodiments, the linker is resistant to cleavage in anextracellular environment. For example, no more than about 20%, no morethan about 15%, no more than about 10%, no more than about 5%, no morethan about 3%, or no more than about 1% of the linkers, in a sample ofconjugate, are cleaved when the conjugate is present in an extracellularenvironment (for example, in plasma). Whether or not a linker isresistant to cleavage in an extracellular environment can be determined,for example, by incubating the conjugate containing the linker ofinterest with plasma for a predetermined time period (for example, 2, 4,8, 16, or 24 hours) and then quantitating the amount of free effectormolecule or detectable marker present in the plasma. A variety ofexemplary linkers that can be used in conjugates are described in WO2004-010957, U.S. Publication No. 2006/0074008, U.S. Publication No.20050238649, and U.S. Publication No. 2006/0024317, each of which isincorporated by reference herein in its entirety.

In several embodiments, conjugates of a CAR, a T cell expressing a CAR,an antibody, or antigen binding portion thereof, and one or more smallmolecule toxins, such as a calicheamicin, maytansinoids, dolastatins,auristatins, a trichothecene, and CC1065, and the derivatives of thesetoxins that have toxin activity, are provided.

Maytansine compounds suitable for use as maytansinoid toxin moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al., PNAS 2002, 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, each of which is incorporated herein by reference. Conjugatescontaining maytansinoids, methods of making same, and their therapeuticuse are disclosed, for example, in U.S. Pat. Nos. 5,208,020; 5,416,064;6,441,163 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference.

Additional toxins can be employed with a CAR, a T cell expressing a CAR,an antibody, or antigen binding portion thereof. Exemplary toxinsinclude Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin andsubunits thereof, ribotoxin, ribonuclease, saporin, and calicheamicin,as well as botulinum toxins A through F. These toxins are well known inthe art and many are readily available from commercial sources (forexample, Sigma Chemical Company, St. Louis, Mo.). Contemplated toxinsalso include variants of the toxins (see, for example, see, U.S. Pat.Nos. 5,079,163 and 4,689,401).

Saporin is a toxin derived from Saponaria officinalis that disruptsprotein synthesis by inactivating the 60S portion of the ribosomalcomplex (Stirpe et al., Bio/Technology, 10:405-412, 1992). However, thetoxin has no mechanism for specific entry into cells, and thereforerequires conjugation to an antibody or antigen binding fragment thatrecognizes a cell-surface protein that is internalized in order to beefficiently taken up by cells.

Diphtheria toxin is isolated from Corynebacterium diphtheriae.Typically, diphtheria toxin for use in immunotoxins is mutated to reduceor to eliminate non-specific toxicity. A mutant known as CRM107, whichhas full enzymatic activity but markedly reduced non-specific toxicity,has been known since the 1970's (Laird and Groman, J. Virol. 19:220,1976), and has been used in human clinical trials. See, U.S. Pat. Nos.5,792,458 and 5,208,021.

Ricin is the lectin RCA60 from Ricinus communis (Castor bean). Forexamples of ricin, see, U.S. Pat. Nos. 5,079,163 and 4,689,401. Ricinuscommunis agglutinin (RCA) occurs in two forms designated RCA60 andRCA120 according to their molecular weights of approximately 65 and 120kD, respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta266:543, 1972). The A chain is responsible for inactivating proteinsynthesis and killing cells. The B chain binds ricin to cell-surfacegalactose residues and facilitates transport of the A chain into thecytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Pat. No.3,060,165).

Ribonucleases have also been conjugated to targeting molecules for useas immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999).Exemplary ribotoxins such as α-sarcin and restrictocin are discussed in,for example Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra,Biochem. 345 Pt 2:247-54, 2000. Calicheamicins were first isolated fromMicromonospora echinospora and are members of the enediyne antitumorantibiotic family that cause double strand breaks in DNA that lead toapoptosis (see, for example Lee et al., J. Antibiot. 42:1070-87,1989).The drug is the toxic moiety of an immunotoxin in clinical trials (see,for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

Abrin includes toxic lectins from Abrus precatorius. The toxicprinciples, abrin a, b, c, and d, have a molecular weight of from about63 and 67 kD and are composed of two disulfide-linked polypeptide chainsA and B. The A chain inhibits protein synthesis; the B chain (abrin-b)binds to D-galactose residues (see, Funatsu et al., Agr. Biol. Chem.52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

A CAR, a T cell expressing a CAR, monoclonal antibodies, antigen bindingfragments thereof, specific for one or more of the antigens disclosedherein, can also be conjugated with a detectable marker; for example, adetectable marker capable of detection by ELISA, spectrophotometry, flowcytometry, microscopy or diagnostic imaging techniques (such as computedtomography (CT), computed axial tomography (CAT) scans, magneticresonance imaging (MRI), nuclear magnetic resonance imaging NMRI),magnetic resonance tomography (MTR), ultrasound, fiberoptic examination,and laparoscopic examination). Specific, non-limiting examples ofdetectable markers include fluorophores, chemiluminescent agents,enzymatic linkages, radioactive isotopes and heavy metals or compounds(for example super paramagnetic iron oxide nanocrystals for detection byMRI). For example, useful detectable markers include fluorescentcompounds, including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, Green fluorescent protein (GFP), Yellow fluorescent protein(YFP). A CAR, a T cell expressing a CAR, an antibody, or antigen bindingportion thereof, can also be conjugated with enzymes that are useful fordetection, such as horseradish peroxidase, β-galactosidase, luciferase,alkaline phosphatase, glucose oxidase and the like. When a CAR, a T cellexpressing a CAR, an antibody, or antigen binding portion thereof, isconjugated with a detectable enzyme, it can be detected by addingadditional reagents that the enzyme uses to produce a reaction productthat can be discerned. For example, when the agent horseradishperoxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which is visuallydetectable. A CAR, a T cell expressing a CAR, an antibody, or antigenbinding portion thereof, may also be conjugated with biotin, anddetected through indirect measurement of avidin or streptavidin binding.It should be noted that the avidin itself can be conjugated with anenzyme or a fluorescent label.

A CAR, a T cell expressing a CAR, an antibody, or antigen bindingportion thereof, may be conjugated with a paramagnetic agent, such asgadolinium. Paramagnetic agents such as superparamagnetic iron oxide arealso of use as labels. Antibodies can also be conjugated withlanthanides (such as europium and dysprosium), and manganese. Anantibody or antigen binding fragment may also be labeled with apredetermined polypeptide epitopes recognized by a secondary reporter(such as leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags).

A CAR, a T cell expressing a CAR, an antibody, or antigen bindingportion thereof, can also be conjugated with a radiolabeled amino acid.The radiolabel may be used for both diagnostic and therapeutic purposes.For instance, the radiolabel may be used to detect one or more of theantigens disclosed herein and antigen expressing cells by x-ray,emission spectra, or other diagnostic techniques. Further, theradiolabel may be used therapeutically as a toxin for treatment oftumors in a subject, for example for treatment of a neuroblastoma.Examples of labels for polypeptides include, but are not limited to, thefollowing radioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

Means of detecting such detectable markers are well known to those ofskill in the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted illumination. Enzymaticlabels are typically detected by providing the enzyme with a substrateand detecting the reaction product produced by the action of the enzymeon the substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

D. Nucleotides, Expression, Vectors, and Host Cells

Further provided by an embodiment of the invention is a nucleic acidcomprising a nucleotide sequence encoding any of the CARs, an antibody,or antigen binding portion thereof, described herein (includingfunctional portions and functional variants thereof). The nucleic acidsof the invention may comprise a nucleotide sequence encoding any of theleader sequences, antigen binding domains, transmembrane domains, and/orintracellular T cell signaling domains described herein.

In some embodiments, the nucleotide sequence may be codon-modified.Without being bound to a particular theory, it is believed that codonoptimization of the nucleotide sequence increases the translationefficiency of the mRNA transcripts. Codon optimization of the nucleotidesequence may involve substituting a native codon for another codon thatencodes the same amino acid, but can be translated by tRNA that is morereadily available within a cell, thus increasing translation efficiency.Optimization of the nucleotide sequence may also reduce secondary mRNAstructures that would interfere with translation, thus increasingtranslation efficiency.

In an embodiment of the invention, the nucleic acid may comprise acodon-modified nucleotide sequence that encodes the antigen bindingdomain of the inventive CAR. In another embodiment of the invention, thenucleic acid may comprise a codon-modified nucleotide sequence thatencodes any of the CARs described herein (including functional portionsand functional variants thereof).

“Nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. In some embodiments, the nucleic aciddoes not comprise any insertions, deletions, inversions, and/orsubstitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

A recombinant nucleic acid may be one that has a sequence that is notnaturally occurring or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination is often accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, e.g., by genetic engineering techniques, such as thosedescribed in Sambrook et al., supra. The nucleic acids can beconstructed based on chemical synthesis and/or enzymatic ligationreactions using procedures known in the art. See, for example, Sambrooket al., supra, and Ausubel et al., supra. For example, a nucleic acidcan be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed upon hybridization (e.g., phosphorothioate derivatives andacridine substituted nucleotides). Examples of modified nucleotides thatcan be used to generate the nucleic acids include, but are not limitedto, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asIntegrated DNA Technologies (Coralville, Iowa, USA).

The nucleic acid can comprise any isolated or purified nucleotidesequence which encodes any of the CARs or functional portions orfunctional variants thereof. Alternatively, the nucleotide sequence cancomprise a nucleotide sequence which is degenerate to any of thesequences or a combination of degenerate sequences.

An embodiment also provides an isolated or purified nucleic acidcomprising a nucleotide sequence which is complementary to thenucleotide sequence of any of the nucleic acids described herein or anucleotide sequence which hybridizes under stringent conditions to thenucleotide sequence of any of the nucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditions mayhybridize under high stringency conditions. By “high stringencyconditions” is meant that the nucleotide sequence specificallyhybridizes to a target sequence (the nucleotide sequence of any of thenucleic acids described herein) in an amount that is detectably strongerthan non-specific hybridization. High stringency conditions includeconditions which would distinguish a polynucleotide with an exactcomplementary sequence, or one containing only a few scatteredmismatches from a random sequence that happened to have a few smallregions (e.g., 3-10 bases) that matched the nucleotide sequence. Suchsmall regions of complementarity are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive CARs. It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

Also provided is a nucleic acid comprising a nucleotide sequence that isat least about 70% or more, e.g., about 80%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,or about 99% identical to any of the nucleic acids described herein.

In an embodiment, the nucleic acids can be incorporated into arecombinant expression vector. In this regard, an embodiment providesrecombinant expression vectors comprising any of the nucleic acids. Forpurposes herein, the term “recombinant expression vector” means agenetically-modified oligonucleotide or polynucleotide construct thatpermits the expression of an mRNA, protein, polypeptide, or peptide by ahost cell, when the construct comprises a nucleotide sequence encodingthe mRNA, protein, polypeptide, or peptide, and the vector is contactedwith the cell under conditions sufficient to have the mRNA, protein,polypeptide, or peptide expressed within the cell. The vectors are notnaturally-occurring as a whole.

However, parts of the vectors can be naturally-occurring. Therecombinant expression vectors can comprise any type of nucleotides,including, but not limited to DNA and RNA, which can be single-strandedor double-stranded, synthesized or obtained in part from naturalsources, and which can contain natural, non-natural or alterednucleotides. The recombinant expression vectors can comprisenaturally-occurring or non-naturally-occurring internucleotide linkages,or both types of linkages. Preferably, the non-naturally occurring oraltered nucleotides or internucleotide linkages do not hinder thetranscription or replication of the vector.

In an embodiment, the recombinant expression vector can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host cell. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescriptseries (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison,Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEXseries (Clontech, Palo Alto, Calif.).

Bacteriophage vectors, such as λ{umlaut over (υ)}TIO, λ{umlaut over(υ)}TI1, λZapII (Stratagene), EMBL4, and λNMI 149, also can be used.Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl 0.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinantexpression vector may be a viral vector, e.g., a retroviral vector or alentiviral vector. A lentiviral vector is a vector derived from at leasta portion of a lentivirus genome, including especially aself-inactivating lentiviral vector as provided in Milone et al., Mol.Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors thatmay be used in the clinic, include, for example, and not by way oflimitation, the LENTIVECTOR® gene delivery technology from OxfordBioMedica plc, the LENTIMAX™ vector system from Lentigen and the like.Nonclinical types of lentiviral vectors are also available and would beknown to one skilled in the art.

A number of transfection techniques are generally known in the art (see,e.g., Graham et al., Virology, 52: 456-467 (1973); Sambrook et al.,supra; Davis et al., Basic Methods in Molecular Biology, Elsevier(1986); and Chu et al, Gene, 13: 97 (1981).

Transfection methods include calcium phosphate co-precipitation (see,e.g., Graham et al., supra), direct micro injection into cultured cells(see, e.g., Capecchi, Cell, 22: 479-488 (1980)), electroporation (see,e.g., Shigekawa et al., BioTechniques, 6: 742-751 (1988)), liposomemediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid mediated transduction (see, e.g., Feigner et al.,Proc. Natl. Acad. Sci. USA, 84: 7413-7417 (1987)), and nucleic aciddelivery using high velocity microprojectiles (see, e.g., Klein et al,Nature, 327: 70-73 (1987)).

In an embodiment, the recombinant expression vectors can be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColEl, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

The recombinant expression vector may comprise regulatory sequences,such as transcription and translation initiation and termination codons,which are specific to the type of host cell (e.g., bacterium, fungus,plant, or animal) into which the vector is to be introduced, asappropriate, and taking into consideration whether the vector is DNA- orRNA-based. The recombinant expression vector may comprise restrictionsites to facilitate cloning.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected host cells.Marker genes include biocide resistance, e.g., resistance toantibiotics, heavy metals, etc., complementation in an auxotrophic hostto provide prototrophy, and the like. Suitable marker genes for theinventive expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding the CAR(including functional portions and functional variants thereof), or tothe nucleotide sequence which is complementary to or which hybridizes tothe nucleotide sequence encoding the CAR. The selection of promoters,e.g., strong, weak, inducible, tissue-specific anddevelopmental-specific, is within the ordinary skill of the artisan.Similarly, the combining of a nucleotide sequence with a promoter isalso within the skill of the artisan. The promoter can be a non-viralpromoter or a viral promoter, e.g., a murine stem cell virus (MSCV)promoter, an Elongation Factor 1 alpha (EF1α) promoter, acytomegalovirus (CMV) promoter, a SV40 promoter, or a RSV promoter. Therecombinant expression vectors can be designed for either transientexpression, for stable expression, or for both. Also, the recombinantexpression vectors can be made for constitutive expression or forinducible expression.

Further, the recombinant expression vectors can be made to include asuicide gene. As used herein, the term “suicide gene” refers to a genethat causes the cell expressing the suicide gene to die. The suicidegene can be a gene that confers sensitivity to an agent, e.g., a drug,upon the cell in which the gene is expressed, and causes the cell to diewhen the cell is contacted with or exposed to the agent. Suicide genesare known in the art (see, for example, Suicide Gene Therapy: Methodsand Reviews, Springer, Caroline J. (Cancer Research UK Centre for CancerTherapeutics at the Institute of Cancer Research, Sutton, Surrey, UK),Humana Press, 2004) and include, for example, the Herpes Simplex Virus(HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleosidephosphorylase, and nitroreductase.

An embodiment further provides a host cell comprising any of therecombinant expression vectors described herein. As used herein, theterm “host cell” refers to any type of cell that can contain theinventive recombinant expression vector. The host cell can be aeukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5a E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell may be a prokaryoticcell, e.g., a DH5a cell. For purposes of producing a recombinant CAR,the host cell may be a mammalian cell. The host cell may be a humancell. While the host cell can be of any cell type, can originate fromany type of tissue, and can be of any developmental stage, the host cellmay be a peripheral blood lymphocyte (PBL) or a peripheral bloodmononuclear cell (PBMC). The host cell may be a T cell. The host cellmay be a natural killer cell (NK cell). The host cell may be ahematopoietic stem cell (HSC).

For purposes herein, the T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. The T cell may be a human T cell. The T cell may be a Tcell isolated from a human. The T cell can be any type of T cell and canbe of any developmental stage, including but not limited to, CD4⁺/CD8⁺double positive T cells, CD4⁺ helper T cells, e.g., Th1 and Th2 cells,CD8⁺ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memoryT cells, memory stem cells, i.e. T_(scm), naïve T cells, and the like.

In an embodiment, the CARs as described herein can be used in suitablenon-T cells. Such cells are those with an immune-effector function, suchas, for example, NK cells, and T-like cells generated from pluripotentstem cells.

Also provided by an embodiment is a population of cells comprising atleast one host cell described herein. The population of cells can be aheterogeneous population comprising the host cell comprising any of therecombinant expression vectors described, in addition to at least oneother cell, e.g., a host cell (e.g., a T cell), which does not compriseany of the recombinant expression vectors, or a cell other than a Tcell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, ahepatocyte, an endothelial cell, an epithelial cell, a muscle cell, abrain cell, etc. Alternatively, the population of cells can be asubstantially homogeneous population, in which the population comprisesmainly host cells (e.g., consisting essentially of) comprising therecombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

CARs (including functional portions and variants thereof), nucleicacids, recombinant expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof),can be isolated and/or purified. For example, a purified (or isolated)host cell preparation is one in which the host cell is purer than cellsin their natural environment within the body. Such host cells may beproduced, for example, by standard purification techniques. In someembodiments, a preparation of a host cell is purified such that the hostcell represents at least about 50%, for example at least about 70%, ofthe total cell content of the preparation. For example, the purity canbe at least about 50%, can be greater than about 60%, about 70% or about80%, or can be about 100%.

E. Methods of Treatment

It is contemplated that the CARs disclosed herein can be used in methodsof treating or preventing HIV/AIDS in a mammal. In this regard, anembodiment provides a method of treating or preventing HIV/AIDS in amammal, comprising administering to the mammal the CARs, the nucleicacids, the recombinant expression vectors, the host cells, thepopulation of cells, the antibodies and/or the antigen binding portionsthereof, and/or the pharmaceutical compositions in an amount effectiveto treat or prevent HIV-1 infection and/or AIDS in the mammal.

An embodiment further comprises lymphodepleting the mammal prior toadministering the CARs disclosed herein. Examples of lymphodepletioninclude, but may not be limited to, non-myeloablative lymphodepletingchemotherapy, myeloablative lymphodepleting chemotherapy, total bodyirradiation, etc.

For purposes of the methods, wherein host cells or populations of cellsare administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal. As used herein, allogeneic means any material derived from adifferent animal of the same species as the individual to whom thematerial is introduced. Two or more individuals are said to beallogeneic to one another when the genes at one or more loci are notidentical. In some aspects, allogeneic material from individuals of thesame species may be sufficiently unlike genetically to interactantigenically. As used herein, “autologous” means any material derivedfrom the same individual to whom it is later to be re-introduced intothe individual.

The mammal referred to herein can be any mammal. As used herein, theterm “mammal” refers to any mammal, including, but not limited to,mammals of the order Rodentia, such as mice and hamsters, and mammals ofthe order Logomorpha, such as rabbits. The mammals may be from the orderCarnivora, including Felines (cats) and Canines (dogs). The mammals maybe from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). Themammals may be of the order Primates, Ceboids, or Simoids (monkeys) orof the order Anthropoids (humans and apes). Preferably, the mammal is ahuman.

With respect to the methods, the cancer associated with the HIVinfection can be any cancer, including any of ALL, AML, alveolarrhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer,brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus,anal canal, or anorectum, cancer of the eye, cancer of the intrahepaticbile duct, cancer of the joints, cancer of the neck, gallbladder, orpleura, cancer of the nose, nasal cavity, or middle ear, cancer of theoral cavity, cancer of the vulva, chronic lymphocytic leukemia (CLL),chronic myeloid cancer (CML), colon cancer, esophageal cancer, cervicalcancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neckcancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma,hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquidtumors, liver cancer, lung cancer (e.g., non-small cell lung carcinomaand lung adenocarcinoma), lymphoma, mesothelioma, mastocytoma, melanoma,multiple myeloma, nasopharynx cancer, NHL, B-chronic lymphocyticleukemia, hairy cell leukemia, Burkitt's lymphoma, ovarian cancer,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, solid tumors, synovial sarcoma,gastric cancer, testicular cancer, thyroid cancer, and ureter cancer.

With respect to the methods, the HIV can be of any type (for example,HIV-1 or HIV-2), group, clade, subtype, sub-subtype, and/or circulatingrecombinant form (CRF), including HIV-1 groups M, N, O, and P. WithinHIV-1 group M, the HIV can be of any clade, sub-subtype of a clade,and/or circulating recombinant form including but not limited to clades,A, A1, A2, A3, A4, B, C, D, F, F1, F2, G, H, J, K, and including but notlimited to circulating recombinant form, CRF01 to CRF90 (see, the worldwide web at hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html). WithinHIV-2, the HIV can be of any subtype of non-recombinant or recombinantsubtype including, A, B, C, D, E, F, G and HIV2_CRF01_AB).

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the methodscan provide any amount or any level of treatment or prevention ofHIV/AIDS in a mammal.

Furthermore, the treatment or prevention provided by the method caninclude treatment or prevention of one or more conditions or symptoms ofthe disease, e.g., HIV-1 infection, being treated or prevented. Also,for purposes herein, “prevention” can encompass delaying the onset ofthe disease, or a symptom or condition thereof.

With respect to the methods, the treatment or prevention by the methodcan include or be administered in conjunction with treatment orprevention of one or more conditions or symptoms but not limited to thefollowing found to co-exist with the disease (for example, HIV/AIDSrelated co-morbidities such as, Kaposi sarcoma or acute myeloidleukemia; viral co-infections such as, HIV/HBV or HIV/HCV co-infection).

With respect to the methods, the treatment or prevention by the methodcan be used in conjunction with an allogenic or autologoustransplantation of cells of mammalian origin lacking disease-associatedgenetic factors (for example but not limited to, bone marrowtransplantation of Δ32-CCR5 cells that are resistant to HIV-1 infectionor genetically-modified CXCR4-negative T-cells).

Another embodiment provides a method of detecting the presence ofinfectious disease in a mammal, comprising: (a) contacting a samplecomprising one or more cells from the mammal with the CARs, the nucleicacids, the recombinant expression vectors, the host cells, thepopulation of cells, the antibodies, and/or the antigen binding portionsthereof, or the pharmaceutical compositions, thereby forming a complex,(b) and detecting the complex, wherein detection of the complex isindicative of the presence of infectious disease in the mammal.

The sample may be obtained by any suitable method, e.g., bloodcollection or biopsy. A blood collection or venipuncture is the processof intravenously collecting venous blood from an individual. A biopsy isthe removal of tissue and/or cells from an individual. Such removal maybe to collect tissue and/or cells from the individual in order toperform experimentation on the removed tissue and/or cells. Thisexperimentation may include experiments to determine if the individualhas and/or is suffering from a certain condition or disease-state. Thecondition or disease may be, e.g., HIV/AIDS.

With respect to an embodiment of the method of detecting the presence ofan infectious disease, e.g., HIV-1 infection, in a mammal, the samplecomprising cells of the mammal can be a sample comprising whole cells,lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction. If the sample comprises whole cells, the cells can be anycells of the mammal, e.g., the cells of any organ or tissue, includingblood cells or endothelial cells. The contacting can take place in vitroor in vivo with respect to the mammal.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the CARs disclosed herein, polypeptides,proteins, nucleic acids, recombinant expression vectors, host cells,populations of cells, or antibodies, or antigen binding portionsthereof, described herein, can be labeled with a detectable label suchas, for instance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles) as disclosed supra.

Methods of testing a CAR for the ability to recognize target cells andfor antigen specificity are known in the art. For instance, Clay et al.,J. Immunol, 163: 507-513 (1999), teaches methods of measuring therelease of cytokines (e.g., interferon-γ, granulocyte/monocyte colonystimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-α) orinterleukin 2 (IL-2). In addition, CAR function can be evaluated bymeasurement of cellular cytotoxicity, as described in Zhao et al, J.Immunol, 174: 4415-4423 (2005).

Methods to assay the ability of a CAR to inhibit HIV-1 infection, butare not limited to, are in-vitro assays that measure HIV-1 viralreplication. For instance, an in-vitro PBMC-based assay that uses areplication competent HIV-1 molecular clone that expresses both aheterologous HIV envelope protein and Renilla luciferase as described inEdmonds et al., Virology, 408: 1-13 (2010) can be used to monitor HIV-1viral replication. In this assay, the level of viral infection can bemonitored by measuring luciferase activity of infected PBMCs eithercontacted with an anti-HIV agent and/or with T-cells expressing achimeric antigen receptor such as described herein this invention. Inanother assay, the level of HIV-1 infection can be assayed bydetermining the amount of p24 antigen present within infected cellculture supernatants using an ELISA assay. In another assay, the levelof HIV-1 infection can be assayed using a qualitative or quantitativemolecular-based method that detects the presence or absence of the viralnucleic acids. In another assay, the presence of infectious HIV-1 viruscan be assayed by inoculating a HIV-permissive cell line and monitoringcytopathic changes indicative of viral infection in combination withother quantitative measures of HIV infection.

Another embodiment provides for the use of the CARs, nucleic acids,recombinant expression vectors, host cells, populations of cells,antibodies, or antigen binding portions thereof, and/or pharmaceuticalcompositions of the invention, for the treatment or prevention of aninfectious disease, e.g., HIV-1 infection, in a mammal. The infectiousdisease may be any of the viruses, microbes, and/or parasites describedherein.

Any method of administration can be used for the disclosed therapeuticagents, including local and systemic administration. For example,topical, oral, intravascular such as intravenous, intramuscular,intraperitoneal, intranasal, intradermal, intrathecal and subcutaneousadministration can be used. The particular mode of administration andthe dosage regimen will be selected by the attending clinician, takinginto account the particulars of the case (for example the subject, thedisease, the disease state involved, and whether the treatment isprophylactic). In cases in which more than one agent or composition isbeing administered, one or more routes of administration may be used;for example, an anti-viral agent may be administered orally and anantibody or antigen binding fragment or conjugate or composition may beadministered intravenously. Methods of administration include injectionfor which the CAR, CAR T Cell, conjugates, antibodies, antigen bindingfragments, or compositions are provided in a nontoxic pharmaceuticallyacceptable carrier such as water, saline, Ringer's solution, dextrosesolution, 5% human serum albumin, fixed oils, ethyl oleate, orliposomes. In some embodiments, local administration of the disclosedcompounds can be used, for instance by applying the antibody or antigenbinding fragment to a region of tissue from which an infectious diseaseis present or has been isolated, or a region suspected of being prone orsupport infectious disease development. In some embodiments, sustainedintra-organ (or near-organ) release of the pharmaceutical preparationthat includes a therapeutically effective amount of the antibody orantigen binding fragment may be beneficial. In other examples, theconjugate is applied as an eye drop topically to the cornea, orintravitreally into the eye.

The disclosed therapeutic agents can be formulated in unit dosage formsuitable for individual administration of precise dosages. In addition,the disclosed therapeutic agents may be administered in a single dose orin a multiple dose schedule. A multiple dose schedule is one in which aprimary course of treatment may be with more than one separate dose, forinstance 1-10 doses, followed by other doses given at subsequent timeintervals as needed to maintain or reinforce the action of thecompositions. Treatment can involve daily or multi-daily doses ofcompound(s) over a period of a few days to months, or even years. Thus,the dosage regime will also, at least in part, be determined based onthe particular needs of the subject to be treated and will be dependentupon the judgment of the administering practitioner.

Typical dosages of the antibodies or conjugates can range from about0.01 to about 30 mg/kg, such as from about 0.1 to about 10 mg/kg.

In particular examples, the subject is administered a therapeuticcomposition that includes one or more of the conjugates, antibodies,compositions, CARs, CAR T cells or additional agents, on a multipledaily dosing schedule, such as at least two consecutive days, 10consecutive days, and so forth, for example for a period of weeks,months, or years. In one example, the subject is administered theconjugates, antibodies, compositions or additional agents for a periodof at least 30 days, such as at least 2 months, at least 4 months, atleast 6 months, at least 12 months, at least 24 months, or at least 36months.

In some embodiments, the disclosed methods include providing surgery,radiation therapy, and/or chemotherapeutics to the subject incombination with a disclosed antibody, antigen binding fragment,conjugate, CAR or T cell expressing a CAR (for example, sequentially,substantially simultaneously, or simultaneously). Methods andtherapeutic dosages of such agents and treatments are known to thoseskilled in the art, and can be determined by a skilled clinician.Preparation and dosing schedules for the additional agent may be usedaccording to manufacturer's instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service, (1992) Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md.

In some embodiments, the combination therapy can include administrationof a therapeutically effective amount of an additional HIV inhibitor,immunomodulatory protein, and/or a protein that enhances the function ofa CAR, CAR-T cells, antibodies, antigen binding fragment, or conjugatesdisclosed herein to a subject. Non-limiting examples of additionaltherapeutic agents that can be used with the combination therapy includemicrotubule binding agents, DNA intercalators or cross-linkers, DNAsynthesis inhibitors, DNA and RNA transcription inhibitors, reversetranscription inhibitors, viral protein inhibitors, immunomodulators,antibodies, enzymes, enzyme inhibitors, gene regulators,anti-proliferative agents, and latency reversing agents. These agents(which are administered at a therapeutically effective amount) andtreatments can be used alone or in combination. For example, anysuitable anti-viral or immunomodulatory agent can be administered incombination with the CARs, CAR-T cells, antibodies, antigen bindingfragment, or conjugates disclosed herein. Methods and therapeuticdosages of such agents are known to those skilled in the art, and can bedetermined by a skilled clinician.

Additional anti-viral agents that can be combined with the CAR, CAR-Tcells, antibodies, antigen binding fragment, or conjugates disclosedherein include, but are not limited to, reverse transcriptase inhibitors(for example, nucleoside reverse transcriptase inhibitors (NRTIs) suchas, tenofovir, adefovir, zidovudine, didanosine, zalcitabine, stavudine,lamivudine, abacavir, emtricitabine, entecavir, and apricitabine);non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as,efavirenz, nevirapine, delavirdine, rilpivirine, and etravirine;protease inhibitors (for example, ritonavir, indinavir, amprenavir,atazanavir, darunavir, tipranavir, saquinavir, nelfinavir, lopinavir,and fosamprenavir); and entry or fusion inhibitors (for example,enfuviritide, maraviroc, gp41-derived C-peptides, and gp41-derivedN-peptides); HIV integrase strand transfer inhibitors (for example,raltegravir, dolutegravir, and elvitegravir), and any combinations ofthese anti-viral agents. In some instances, to reverse HIV latency, alatency reversing agent can be combined with the CAR, CAR-T cells,antibodies, antigen binding fragment, or conjugates disclosed herein.Examples of such compounds include, but not limited to, PKC agonists(for example, Bryostatin, Prostratin, Ingenol B), innate immuneactivators (e.g., TLR7 agonist, IL-15SA), histone deacetylase inhibitors(for example, suberoylanilide hydroxamic acid (vorinostat), romidepsin,panobinostat), DNA demethylation agents, and other chromatin remodelingagents. Selection and therapeutic dosages of such agents are known tothose skilled in the art, and can be determined by a skilled clinician.

Additional radiation or chemotherapeutic agents that can be combined totreat, prevent, or synergize with the method include, but are notlimited to alkylating agents, such as nitrogen mustards (for example,chlorambucil, chlormethine, cyclophosphamide, ifosfamide, andmelphalan), nitrosoureas (for example, carmustine, fotemustine,lomustine, and streptozocin), platinum compounds (for example,carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan,dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, anduramustine; antimetabolites, such as folic acid (for example,methotrexate, pemetrexed, and raltitrexed), purine (for example,cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine),pyrimidine (for example, capecitabine), cytarabine, fluorouracil, andgemcitabine; plant alkaloids, such as podophyllum (for example,etoposide, and teniposide), taxane (for example, docetaxel andpaclitaxel), vinca (for example, vinblastine, vincristine, vindesine,and vinorelbine); cytotoxic/antitumor antibiotics, such as anthracyclinefamily members (for example, daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin,hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecanand irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab,cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, andtrastuzumab; photosensitizers, such as aminolevulinic acid, methylaminolevulinate, porfimer sodium, and verteporfin; and other agents,such as alitretinoin, altretamine, amsacrine, anagrelide, arsenictrioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib,celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib,hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin,masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib,vemurafinib, vandetanib, and tretinoin. Selection and therapeuticdosages of such agents are known to those skilled in the art, and can bedetermined by a skilled clinician.

The combination therapy may provide synergy and prove synergistic, thatis, the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation, a synergistic effect maybe attained when the compounds are administered or deliveredsequentially, for example by different injections in separate syringes.In general, during alternation, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

In one embodiment, an effective amount of an CAR, CAR-T cells, antibody,or antigen binding fragment that specifically binds to one or more ofthe antigens disclosed herein or a conjugate thereof is administered toa subject having HIV/AIDS. After a sufficient amount of time has elapsedto allow for the administered antibody or antigen binding fragment orconjugate to form an immune complex with the antigen expressed on therespective HIV-1 infected cell, the immune complex is detected. Thepresence (or absence) of the immune complex indicates the effectivenessof the treatment. For example, an increase in the immune complexcompared to a control taken prior to the treatment indicates that thetreatment is not effective, whereas a decrease in the immune complexcompared to a control taken prior to the treatment indicates that thetreatment is effective.

F. Biopharmaceutical Compositions

Biopharmaceutical or biologics compositions (hereinafter,“compositions”) are provided herein for use in gene therapy,immunotherapy and/or cell therapy that include one or more of thedisclosed CARs, or T cells expressing a CAR, antibodies, antigen bindingfragments, conjugates, CARs, or T cells expressing a CAR thatspecifically bind to one or more antigens disclosed herein, in a carrier(such as a pharmaceutically acceptable carrier). The compositions can beprepared in unit dosage forms for administration to a subject. Theamount and timing of administration are at the discretion of thetreating clinician to achieve the desired outcome. The compositions canbe formulated for systemic (such as intravenous) or local (such asintra-organ) administration. In one example, a disclosed CARs, or Tcells expressing a CAR, antibody, antigen binding fragment, conjugate,is formulated for parenteral administration, such as intravenousadministration. Compositions including a CAR, or T cell expressing aCAR, a conjugate, antibody or antigen binding fragment as disclosedherein are of use, for example, for the treatment and detection of aninfectious disease, for example, and not by way of limitation, a HIV-1infection. The compositions including a CAR, or T cell expressing a CAR,a conjugate, antibody or antigen binding fragment as disclosed hereinare also of use, for example, for the detection of immune dysfunction.

The compositions for administration can include a solution of the CAR,or T cell expressing a CAR, conjugate, antibody or antigen bindingfragment dissolved in a pharmaceutically acceptable carrier, such as anaqueous carrier. A variety of aqueous carriers can be used, for example,buffered saline and the like. These solutions are sterile and generallyfree of undesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, adjuvant agents, and the like, forexample, sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate and the like. The concentration of a CAR, or Tcell expressing a CAR, antibody or antigen binding fragment or conjugatein these formulations can vary widely, and will be selected primarilybased on fluid volumes, viscosities, body weight and the like inaccordance with the particular mode of administration selected and thesubject's needs. Actual methods of preparing such dosage forms for usein gene therapy, immunotherapy and/or cell therapy are known, or will beapparent, to those skilled in the art.

A typical composition for intravenous administration includes about 0.01to about 30 mg/kg of antibody or antigen binding fragment or conjugateper subject per day (or the corresponding dose of a CAR, or T cellexpressing a CAR, conjugate including the antibody or antigen bindingfragment). Actual methods for preparing administrable compositions willbe known or apparent to those skilled in the art and are described inmore detail in such publications as Remington's Pharmaceutical Science,19th ed., Mack Publishing Company, Easton, Pa. (1995).

A CAR, or T cell expressing a CAR, antibodies, antigen bindingfragments, or conjugates may be provided in lyophilized form andrehydrated with sterile water before administration, although they arealso provided in sterile solutions of known concentration. The CARs, orT cells expressing a CAR, antibody or antigen binding fragment orconjugate solution is then added to an infusion bag containing 0.9%sodium chloride, USP, and in some cases administered at a dosage of from0.5 to 15 mg/kg of body weight. Considerable experience is available inthe art in the administration of antibody or antigen binding fragmentand conjugate drugs; for example, antibody drugs have been marketed inthe U.S. since the approval of RITUXAN® in 1997. A CAR, or T cellexpressing a CAR, antibodies, antigen binding fragments and conjugatesthereof can be administered by slow infusion, rather than in anintravenous push or bolus. In one example, a higher loading dose isadministered, with subsequent, maintenance doses being administered at alower level. For example, an initial loading dose of 4 mg/kg antibody orantigen binding fragment (or the corresponding dose of a conjugateincluding the antibody or antigen binding fragment) may be infused overa period of some 90 minutes, followed by weekly maintenance doses for4-8 weeks of 2 mg/kg infused over a 30-minute period if the previousdose was well tolerated.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995). Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin or a drug, as a central core. In microspheres, thetherapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992).

Polymers can be used for ion-controlled release of the CARs, or T cellsexpressing a CAR, antibody or antigen binding fragment or conjugatecompositions disclosed herein. Various degradable and non-degradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known (seeU.S. Pat. Nos. 5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028;4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164;5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496).

G. Kits

In one aspect, kits employing the CARs disclosed herein are alsoprovided. For example, kits for treating HIV/AIDS in a subject, ormaking a CAR T cell that expresses one or more of the CARs disclosedherein. The kits will typically include a disclosed antibody, antigenbinding fragment, conjugate, nucleic acid molecule, CAR or T cellexpressing a CAR as disclosed herein. More than one of the disclosedantibodies, antigen binding fragments, conjugates, nucleic acidmolecules, CARs or T cells expressing a CAR can be included in the kit.

The kit can include a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container typicallyholds a composition including one or more of the disclosed antibodies,antigen binding fragments, conjugates, nucleic acid molecules, CARs or Tcells expressing a CAR. In several embodiments the container may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). A label or package insert indicates that thecomposition is used for treating the particular condition.

The label or package insert typically will further include instructionsfor use of a disclosed antibodies, antigen binding fragments,conjugates, nucleic acid molecules, CARs or T cells expressing a CAR,for example, in a method of treating or preventing HIV/AIDS or of makinga CAR T cell. The package insert typically includes instructionscustomarily included in commercial packages of therapeutic products thatcontain information about the indications, usage, dosage,administration, contraindications and/or warnings concerning the use ofsuch therapeutic products. The instructional materials may be written,in an electronic form (such as a computer diskette or compact disk) ormay be visual (such as video files). The kits may also includeadditional components to facilitate the particular application for whichthe kit is designed. Thus, for example, the kit may additionally containmeans of detecting a label (such as enzyme substrates for enzymaticlabels, filter sets to detect fluorescent labels, appropriate secondarylabels such as a secondary antibody, or the like). The kits mayadditionally include buffers and other reagents routinely used for thepractice of a particular method. Such kits and appropriate contents arewell known to those of skill in the art.

EXAMPLES

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

Example 1

Assembly of HIV-Specific Binders into Functional CAR Molecules

This example describes general methods for constructing monospecific,bispecific, and trispecific anti-HIV CARs containing the mD1.22, m36.4,and C46 peptide as well how to express these CARs on the surface ofprimary T-cells.

Materials and Methods:

Generation of Lentiviral Vector Constructs

CAR antigen-binding domain sequences were derived from publishedsequences (Chen et al., J. Virol. 2014; 88:(2) 1125-1139; Chen et al.,Antiviral Research 2010; 88:(1) 107-115; Egerer et al., MolecularTherapy 2010; 19:(7) 1236-1244) and synthesized by ATUM (formerly knownas, DNA 2.0; Newark, Calif.) or IDT Technologies (Coralville, Iowa).Synthesized gene fragments were subcloned in-frame to a MSCVpromoter-based lentiviral backbone containing the CD8 linker/hinge, CD8transmembrane domain, 41-BB, and CD3ζ signaling domain. For bicistronicbispecific and trispecific CAR constructs a cleavable furin-P2A-furinsite was placed downstream of the first CAR containing a single ortwo-linked antigen binding domains followed by third antigen bindingdomain, CD8 linker/hinge, TNFRSF19 transmembrane, and with or without aCD3 signaling domain. A detailed description of the sequence and CARstructure is shown below in the Table 1. The DNA constructs wereconfirmed by sanger sequencing (GeneWiz, South Plainfield, N.J.).Plasmid maps were generated using Clone Manager software (Denver,Colo.).

Lentiviral Vector Production

Lentiviral vectors carrying a CAR transgene were produced by transientlytransfecting 293T suspension cells using a four-plasmid system in thepresence of polyethylenimine (PEI). Briefly, suspension 293T wereco-transfected with the CAR transfer plasmid, VSVg envelope, gag/pol,and rev plasmids followed by addition of sodium butyrate to cultures 16hours later. Supernatants (˜400 mL) containing the lentiviral vectorswere concentrated 48 hours later by overnight ultracentrifugation at10,000×g for at least 18 hours. Pellets were resuspended in 2 mL offiltered-sterile SEC buffer containing 5.96 mM HEPES, 5% Trehalose, and100 mM NaCl until the pellet was easy to dissolve by gentle agitation.Resuspended lentiviral particles were stored in aliquots at −80° C.until further use.

Anti-HIV CAR Detection on the Surface of Transduced Primary T-Cells

Approximately 1×106 cells were washed in MACS buffer (Phosphate-bufferedsaline containing 10% bovine serum albumin, pH˜7.2). Vioblue-conjugatedCD4 (clone VIT4) and FITC-labeled CD8 antibodies were then added to thecells following the manufacturer's instructions (Miltenyi Biotec). Aftera 30-minute incubation at 4° C., cells were washed twice in MACS bufferand resuspended in 0.2 mL of MACS buffer. For detection of CARscontaining the C46 peptide, the recombinant human monoclonal antibody2F5 (Polymun Scientific. Klosterneuburg, Austria) was added at adilution of 1:1000 for 30 minutes at 4° C. followed by washing twice inMACS buffer. 2F5 recognizes the epitope ELDKWA found within the C46peptide. Cells were then incubated with FITC-labeled F(ab′)2 anti-humanIgG for 30 minutes at 4° C., washed twice, and then resuspended in 0.2mL MACS buffer. Flow cytometry was carried out using a MACS Quant VYB1cytometer (Miltenyi Biotec) and analysis was performed using FlowJosoftware (Tree Star, Ashland, Oreg.).

Results:

Overall, anti-HIV CARs were highly expressed on the surface of primaryT-cells ranging up to 70% gene modification using lentiviral vectors. Asshown in FIGS. 3A and 3B, monospecific CARs containing either the mD1.22domain (LTG1944), m36.4 domain (LTG1945), or C46 peptide (LTG2328) werefunctionally-expressed on the surface of T-cells. The m36.4 CAR wasdetected indirectly by fusing an intracellular mCherry reporter upstreamof the CD3 signaling domain (FIG. 3B).

To define the optimal bispecific binder architecture of CARs configuredwith the mD1.22 and m36.4 domains, up to seven different bispecific CARconstructs were constructed. These CARs were engineered in variousorientations and using different linker lengths to preserved domainmodularity and functionality. As compared to untransduced T-cells,bispecific CARs containing the shortest glycine-serine linker tospatially separate the mD1.22 and m36.4 domains (LTG2325, a single G45motif) or up to the longest linker (LTG1947, five G45 motifs) wereequally expressed with similar transduction efficiencies (FIG. 4A).Reversing the orientation of the two domains to present m36.4 distal tomD1.22 (LTG1948) only slightly reduced CAR transduction efficiency (FIG.4B). This is most likely attributed to epitope accessibility of the CD4antibody rather than to construct design. To allow for maximal functionof the two domains, a bicistronic P2A construct was generated containingtwo CARs, mD1.22-CAR and m36.4-CAR, to form LTG2303. The rationale forthis construct is that m36.4 alone can bind to and neutralize HIV-1strains albeit with reduced affinity in the absence of the CD4 receptor(Weizao Chen et al., Journal of Virology 2014, 88:2 1125-1139). Thus,engineering both domains as a CAR would improve CAR-mediatedcytotoxicity. As shown in FIG. 4C, the mD1.22-CAR portion of the LTG2303bicistronic construct was detected on the surface of T-cells. Furtherevaluation of this construct using anti-CD3 western blot revealed thatboth CARs were highly expressed, fully cleaved, and migrated to theirpredicted molecular weight (FIG. 4D). Overall, bispecific CARs werehighly expressed on the surface of T-cells with similar transductionefficiencies across different constructs (FIG. 4E).

Next, trispecific CARs containing all three domains (mD1.22, m36.4, andC46 peptide) were generated using the LTG1946 and LTG2303 architectures.Specifically, trispecific CARs were engineered to generate (1) CARs thatcontain all three domains on a single CD3 (LTG2318, LTG2319, andLTG2320), (2) CARs that contain the LTG1946 or LTG1947 bispecific CAR incombination with the C46 peptide anchored to the T-cell membrane alone(LTG2323 and LTG2334), and lastly (3) CARs that contain the LTG2303bispecific CAR with C46 peptide placed distal to the mD1.22 domain(LTG2329 and LTG2330). All these combinations form unique trispecificCARs to effectively interrogate trispecific CAR function.

As shown in FIG. 11A, trispecific CARs containing all three domains on asingle CD3 chain were highly expressed (˜50-70%) as detected by 2F5 flowcytometry (LTG2318, LTG2319, LTG2320). In contrast, detection of themembrane anchored C46 peptide in the bicistronic trispecific construct(LTG2323) was markedly reduced (FIG. 11A, 18%). However, the mD1.22-CARportion of the LTG2323 trispecific construct was highly expressed on thesurface of T-cells as compared to untransduced T-cells (FIG. 11B).Similarly, trispecific CARs LTG2329 and LTG2330 were detectable by 2F5flow cytometry (up to 40%, FIG. 12C). Taken together, trispecific CARsare functionally-detected on the surface of T-cells.

TABLE 1 List of HIV-Targeting CAR Constructs LV Construct CAR StructureLTG1944 LP-mD1.22-CD8TM-41BB-CD3zeta LTG1945 LP-m36.4-CD8TM-41BB-CD3zetaLTG2328 LP-C46-CD8TM-41BB-CD3zeta LTG2325LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta LTG2313LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta LTG1946LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta LTG2326LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta LTG1947LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta LTG1948LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3zeta LTG2303LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2- m36.4-TNFRSF19TM-CD3zeta2 LTG2322LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2- m36.4-TNFRSF19TM LTG2314LP-mD1.22-L3-C46-CD8TM-41BB-CD3zeta LTG2315LP-mD1.22-L5-C46-CD8TM-41BB-CD3zeta LTG2316LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta LTG2317LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta LTG2318LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB- CD3zeta LTG2319LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB- CD3zeta LTG2320LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB- CD3zeta LTG2323LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta- F2AF-LP2-C46-TNFRSF19TM LTG2329LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF- LP2-m36.4-TNFRSF19TM-CD3zeta2LTG2330 LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2 LTG2331LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF- LP2-m36.4-TNFRSF19TM LTG2332LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF- LP2-m36.4-TNFRSF19TM LTG2334LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF- LP2-C46-TNFRSF19TM

Example 2

Novel Bispecific and Trispecific Anti-HIVCARs Potently DestroyHIV-Envelope Targets

This Example describes the functional characterization of anti-HIV CARsas determined by a highly sensitive luciferase-based cytotoxicity assay.In addition, activation of T-cells is determined by quantifying cytokinesecretion in the absence and presence of HIV-envelope expressing targetcells.

Materials and Methods:

Cell Lines Used for Functional Characterization

The 293T cell line was engineered to stably expressing the single-chainfull-length HIV envelope protein (293T-Env) and was kindly provided byDr. Dimiter Dimitrov (NCI, Fort Detrick, Md.). Briefly, 293T-Env cellswere grown in Dulbecco's modified eagle medium (DMEM) in the presence of10% fetal bovine serum and 60 μg/ml of zeocin to maintain selection. Togenerate luciferase-expressing cells, 293T-Env cells were transducedwith a lentiviral vector containing the firefly luciferase gene, singlecell cloned, and tested for both gp120/gp41 expression on the surface of293T cells (via 2G12, b12, and 2F5 flow cytometry) and luciferaseactivity. A single high-expressing HIV-1 envelope and luciferase clonewas isolated and used for cytotoxicity assays described herein(293T-Env-Luc). For envelope-negative cell lines, Raji and 293T HEKcells were purchased from ATCC (Manassas, Va.) and subsequentlytransduced with a lentiviral vector encoding the firefly luciferase geneto generate Raji-Luc or 293T-Luc cell lines, respectively. Raji-Luccells were maintained in RPMI media containing 10% fetal bovine serum.293T-Luc cells were maintained in DMEM media containing 10% fetal bovineserum. Both cell lines were single-cell cloned and the absence ofHIV-envelope confirmed by 2G12, b12, and 2F5 (monoclonal antibodiesagainst HIV-1 envelope) flow cytometric analysis.

Primary T Cell Purification and Transduction

Human PBMCs from healthy donors were purified from buffy coats usingficoll-paque gradient separation combined with a leucosep tube followedby immunomagnetic bead selection of CD4⁺ and CD8⁺ T-cells according tomanufacturer's protocol (Miltenyi Biotec, Bergisch-Gladbach, Germany).On day 0, T cells were activated with CD3/CD28 MACS® Large-scale T CellTransAct reagent (Miltenyi Biotec) in the presence of 40 IU/mL IL-2(Miltenyi Biotec). On day 3, activated T-cells were transduced withlentiviral vectors encoding CAR constructs in the presence of 10 μg/mprotamine sulfate (Sigma-Aldrich, St. Louis, Mo.) and 200 IU/mL IL-2.Cultures were propagated in TexMACS medium supplemented with 200 IU/mlIL-2 until harvest on day 9-10 for functional analysis.

Cytotoxicity Assay Using Luciferase Detection

Briefly, 5×103 target cells stably expressing firefly luciferase wascombined with or without CAR T-cells at various effector to targetratios in a sterile 96-well plate and incubated overnight at 37° C. inthe presence of 5% CO2. Twenty-four hours later, 100 μL of SteadyGloreagent (Promega, Madison Wis.) was added to each well and incubated for10 minutes at room temperature followed by quantification ofluminescence using an Enspire plate reader (Perkin Elmer, Waltham,Mass.). The luminescence was captured as counts per second (CPS) foreach experimental well containing the indicated E:T ratio (sample CPS)and target cells alone (target CPS). Percent specific lysis wascalculated as: 1−(sample CPS/target CPS).

Cytokine Release Assay Using Sandwich ELISA

Supernatants from the cytotoxicity assay were collected (E:T ratio of10:1), diluted 10-fold, and assayed for IFN-γ and IL-2 by Ready-Set-GoELISA as per the manufacturer's instructions (eBioscience, San Diego,Calif.).

Results:

The HIV envelope protein is a heterotrimeric glycoprotein expressed onthe surface of the HIV virion which is used to target and hijackT-cells. During HIV infection, the HIV envelope protein (gp120) binds tothe CD4 receptor of helper T-cells leading to a conformation change inthe HIV envelope protein. This change promotes its interaction withco-receptors (e.g., CCR5 or CXCR4) on the surface of T-cells in atropic-dependent manner ultimately culminating in viral fusion andsubsequent intracellular release of the HIV viral RNA to the T-cell. Theviral RNA is subsequently reverse transcribed and integrated into thehost genome in which it can produce more infectious virus or remain in alatent state. When T-cells are actively producing HIV virus, a portionof the HIV envelope protein (gp120/gp41) remains on the surface of thoseinfected T-cells as a consequence of viral budding. Hence, theproductive HIV-infected cell can be specifically targeted by a CARengineered to recognize the HIV envelope glycoprotein followed byelimination of those HIV-infected cells by virtue of its design.

HIV envelope epitopes have been used as targets for passiveimmunotherapy with monoclonal antibodies, but without broad success(reviewed in Mascola J R, Haynes B F. Immunological reviews. 2013;254(1):225-244; Jaworski J P, Vendrell A, Chiavenna S M. Frontiers inImmunology. 2016; 7:661). In the absence of ART, antibodies alone leadto rapid viral rebound and ultimately viral escape. This loss ofvirologic control is largely due to antibody instability and a gradualdecline of antibody concentrations in the host. Hence, HIV therapiesthat can provide a durable response are more likely to not only controlHIV in the absence of ART but possibly lead to eradication of the HIVreservoir.

With the success of the CD19 CAR, revisiting CAR-based strategies totreat and possibly cure HIV is a highly attractive approach. Whencombined with novel anti-HIV domains and state-of-art genome engineeringtools (for example, lentiviral vectors), CARs may address the limitationof passive immunization strategies and overcome previous pitfalls in HIVCAR design. More importantly, CARs can be integrated with a new “kickand kill” paradigm that focuses on eliminating the HIV reservoir viacontinuous immunosurveillance and specialized drugs that reawaken thelatent HIV.

Herein described are a unique collection of anti-HIV CARs that exertpotent cytotoxic and cytokine functions against envelope-positive cellswhile maintaining exceptional specificity. As shown below in Table 1,more than 20 anti-HIV CARs were designed to fully interrogate theirpotency against HIV-1. These CARs were designed by uniquely combiningthree different HIV envelope binding sequences in a precise way to bepotent and synergistic inhibitors of HIV-1 infection. Functionalcharacterization of these binders as CARs and iterations thereof weregenerated by cloning into lentiviral expression vectors that containedselected structural and signaling domains under the control of aconstitutively active promoter and tested in vitro for transductionefficiency, killing function, and cytokine production using aHIV-envelope cell line model. Table 1 summarizes the nomenclature used.In some experiments CAR construct LTG1732, a mCherry reporter, serves asa negative control to evaluate changes in T-cell function as a result ofviral transduction.

In the early 2000s, a CD4-zeta CAR entered into clinical trials.Although these studies demonstrated a lack of efficacy, these CARspersisted for decades. Possible explanations for the lack of virologiccontrol argue that the initial first generation CD4ζ CAR requiresre-engineering using the current understanding of CARs. Hence, andsimilar to others in the CAR field, a CD4-CAR was designed andengineered with the second-generation structural elements describedherein and compared to the more potent, specific, and compactCD4-derived mD1.22-CAR (LTG1944) disclosed herein. While the mD1.22-CARwas potent, in contrast to the CD4-CAR, the mD1.22-CAR demonstratedimproved specificity elaborating a trend of lower levels of off-targetcytokines (data not shown). Thus, the novel CD4-like yet improvedmD1.22-CAR described supra was selected as the initial prototype forfurther iterative CAR design.

To fully understand the relative contribution of each binder in thecontext of a CAR, the m36.4-CAR (LTG1945) and C46-CAR (LTG2328) wasfurther designed and evaluated. As shown in FIG. 3C, mD1.22-CAR(LTG1944) was the most potent followed by m36.4-CAR (LTG1945) andC46-CAR (LTG2328). All three CARs demonstrated exceptional specificitywith no off target killing in the absence of HIV-envelope (FIG. 3D).Moreover, these CARs with the exception of C46-CAR were specificallytriggered by HIV-envelope expressing cells to secrete IFN-γ (FIG. 3E), amarker of antigen-driven T-cell activation. Taken together, themD1.22-CAR was the most optimal architecture for building more advancedbi- and tri-specific HIV CARs.

To engineer a bispecific HIV CAR, an iterative approach was employed todetermine the best way to combine mD1.22 and m36.4 for optimal CARfunction. As shown in FIG. 5, all bispecific CARs destroyed up to 80% ofenvelope-expressing targets (FIG. 5) and were highly specific (FIG. 6).However, T-cells engineered with both a mD1.22-CAR and a m36.4-CAR usinga bicistronic P2A vector (LTG2303) were significantly more potent thancells only expressing a single CAR such as, LTG1944 or LTG1945 asmeasured by cytotoxicity (FIG. 5) and cytokine release (FIG. 7).Reversing the order of the two domains to place m36.4 distal to mD1.22had no significant effect on CAR activity (LTG1948). As demonstrated inFIG. 8A, decreasing the spatial distance between the mD1.22 and m36.4domains to a single G4S motif significantly enhanced CAR-mediatedcytotoxicity (LTG2325 versus LTG1947). Moreover, these linker-specificCARs exerted no cytotoxicity on envelope-negative 293T or Raji cells(FIG. 8B). Upon further interrogation of these linker-specific CARs, allanti-HIV CARs were specifically-triggered to produce IFN-γ and in someinstances increased potency was often accompanied by increasedoff-target IFN-γ production (FIG. 9). Overall, a series of functionalassays identified LTG2303 as the most potent bispecific CAR architecturefor further trispecific HIV CAR design.

A trispecific HIV CAR architecture was then designed and evaluated whichstarted with the basic mD1.22-CAR architecture and designed severalintermediate constructs to evaluate the optimal placement of a highlypotent C-peptide, C46. C-peptides (for example, T20 or C46) are derivedfrom highly conserved regions within the C-terminus heptad repeat (CHR)region of gp41. In their native state, hydrophobic interactions betweenC-peptides and the N-terminus heptad repeat region (NHR) are involved inviral fusion. Interestingly, several unique strategies using C-peptidesas decoys have clearly demonstrated their ability to potently inhibitviral fusion leading to suppressed HIV-1 infection. For instance, a fewpublished strategies show that anchoring the C-peptide to the T-cellmembrane potently ablates HIV-1 infection (van Lunzen et al., MolecularTherapy 2007, 15: (5) 1024-1033; Kimpel et al., PLOS One 2010, 5:(8)e12357; Melikyan et al. Journal of Virology 2006, 80:(7) 3249-3258). Ina second strategy, enabling T-cells to constitutively secrete aC-peptide (SAVE) also protected bystander T-cells from HIV-1 infection(Egerer et al., Molecular Therapy 2010, 19(7), 1236-1244). In 2003,Enfuviritide (T20) was approved by the FDA as a salvage therapy fortreating patients with multidrug resistant HIV-1. However emerging T20resistant mutants have led to development of improved C-peptide design.

These new C-peptides such as C46 have proven to inhibit T20 resistantHIV-1 mutants. Similar to other C-peptides, C46 targets an extended,overlapping T20 site also involved in gp41-mediated viral fusion.Moreover, improved C-peptides have been found to inhibit hard toneutralize HIV-1 strains independent of viral tropism and exhibitsimproved breadth alone and in combination with mD1.22-based antibodies(Yao et al., The Journal of Biological Chemistry 2012, 287:(9),6788-6796; Qi et al., Emerging Microbes & Infections 2017, 6:(6)).Hence, it was rationalized that engineering a trispecific CAR with theC46 peptide would be an attractive strategy to protect T-cells fromHIV-1 infection as well as serve to enhance breadth of CARs uponencountering highly-resistant HIV-1 viruses.

As shown in FIG. 10, C46-based CARs were configuration-dependent andfunctioned best when engineered with the C46 peptide oriented distal tothe mD1.22 domain (LTG2316, LTG2317 versus LTG2314, LTG2315). Aspreviously shown using m36.4-based bispecific CARs, increasing thelinker length between the C46 and mD1.22 domains also led to a reductionin cytotoxicity (FIG. 10A, LTG2316 versus LTG2317). However, thisslightly less active CAR (LTG2317) demonstrated improved specificity asmeasured by IFN-γ secretion (FIG. 10B). Furthermore, engineering themD1.22-CAR with either the m36.4 domain (LTG1946) or the C46 peptide(LTG2316) exerted similar cytotoxic effects and released IFN-γ in thepresence of envelope-expressing targets (FIGS. 10A and B). Inconclusion, an iterative approach was used to determine the optimalbinder architecture for bispecific HIV CARs. This approach revealed aset of rules that govern HIV CAR design and identified two highly potentCAR architectures to serve as the prototype for trispecific HIV CARdesign.

Herein, trispecific anti-HIV CARs are described and characterized bytheir cytotoxic capabilities. Three unique trispecific CAR architectureswere engineered by (1) fusing all three domains together on a single CD3(LTG2318, LTG2319, LTG2320), or by employing the (2) bispecific CARLTG1946 combined with a membrane-anchored C46 peptide (LTG2323,LTG2334), and lastly by employing the (3) bispecific CAR LTG2303combined with C46 peptide placed distal to the mD1.22 domain (LTG2329,LTG2330). As described in Example 1, these trispecific CARs wereexpressed on the surface of primary T-cells. Functionally, trispecificCARs performed as well as bispecific CARs using the surrogateHIV-envelope expressing cell line with modest off-target killing at veryhigh effector to target ratios. As shown in FIG. 11C, trispecific CARsengineered with all three domains on a single CD3ζchain were highlyinfluenced by the location of the C46. For instance, placing the C46peptide between the mD1.22 domain and m36.4 domain (LTG2319) or near theT-cell membrane (LTG2318) abrogated its cytolytic function. However,presenting the C46 domain first followed by the other two domains(LTG2320) or anchoring the C46 peptide to the T-cell membraneindependent of the CAR (LTG2323) led to robust cytolytic function andsimilar function to bispecific CAR LTG1946 (FIG. 11C). These trispecificCARs were highly specific with no off-target killing onenvelope-negative cells (FIG. 11D) and they produced cytokines inresponse to antigen (FIGS. 11 E-F). Among the first set of trispecificCARs, LTG2323 appeared to be the most potent.

Similarly, the third set of trispecific CARs LTG2329 and LTG2330 alsodemonstrated robust cytolytic effect (FIG. 12B) with some increase innon-specific killing at very high E:T ratios (FIG. 12D). Thisobservation was only found in T-cells co-expressing two different CARs(LTG2303, LTG2329, and LTG2330). As previously observed with bispecificCARs (LTG2316 versus LTG2317), increasing the linker length also reducedoff-target cytotoxicity (FIG. 12D, LTG2330 versus LTG2329). Hence,further optimization of the CAR architecture may improve CARspecificity. In conclusion, bispecific and trispecific anti-HIV CARs arehighly potent and represent a novel class of therapies to treatHIV/AIDS.

Example 3

Bispecific and Trispecific Anti-HIV duoCAR-T Cells Broadly and PotentlyEliminate HIV-Infected PBMC In Vitro and In Vivo

This Example interrogates anti-HIV CAR-T cell killing efficacy upon invitro and in vivo challenge with PBMC infected with differentEnv-IMC-LucR HIV-1 viruses as well as the susceptibility of CAR-T cellsto HIV-1 infection.

Materials and Methods:

In Vitro Efficacy of Anti-HIV CARs Using Replication-CompetentEnv-IMC-LucR Molecular Clones to Infect Donor Matched PBMC

Inhibition of HIV-1 infection was investigated using areplication-competent HIV-1 molecular clone that contains a desiredheterologous HIV-1 envelope upstream of an in-frame Renilla luciferaseORF (Env-IMC-LucR). The infectious clones were generated as previouslydescribed (Edmonds et al., Virology 2010, 408: 1-13). Upon HIV-1infection of PBMCs or CD4⁺ T-cells, the expression of Renilla luciferaseserves as a highly sensitive and quantifiable measure of HIV-1 viralreplication up to several weeks post-inoculation. Briefly, autologousdonor PBMCs (HIV-1 naïve) were activated using PHA (4 μg/mL) and IL-2(100 U/mL) and cultured at 37° C. in R10 media (RPMI supplemented with10% heat-inactivated FBS, penicillin (100 U/mL), streptomycin (10μg/mL), glutamine (2 mM), and HEPES (10 mM)). One day later,1×10{circumflex over ( )}5 PBMCs were spinfected with 1×10{circumflexover ( )}5 IU/mL (MOI=1) of the indicated Env-IMC-LucR virus in a 96well round-bottom plate for 24 hours. The following day, 1×10{circumflexover ( )}5 effector cells (anti-HIV CAR T cells) were added to theinfected PBMCs and co-cultured for 7 days. For in vitro protectionassays, anti-HIV CAR-T effectors were directly challenged with theindicated Env-IMC-LucR virus at a MOI=1 in the absence of PBMCs. Cellculture supernatants (60 μL) were taken from co-cultures on days 0, 3,5, and 7 for further analysis and cultures replenished with R10 media.After 7 days, cells were pelleted and lysed with 20 μl of lysis bufferand luminescence quantified (relative light units, RLU) as permanufacturer's instructions (Promega, Madison, Wis.). Data was generatedusing three independent donors (error bars=standard deviation).Luciferase activity was quantified following the manufacturer'sinstructions. The data was presented as relative light units (RLU). Forin vitro killing assays, the log inhibition of HIV-1 infection wascalculated by the following formula: Log inhibition of HIV-1infection=Log 10(CARRLU/UTDRLU). Percent HIV-1 inhibition was calculatedby 1−(CARRLU/UTDRLU)×100%. Statistical analysis was performed using amultiple analysis student t-test for in vitro killing efficacy andone-way ANOVA for in vitro protection assays.

Multi-Specific Anti-HIV duoCAR T Cells Potently Eliminate Acute andChronically HIV-Infected PBMC within a Humanized NSG Mouse Model

The in vivo efficacy of anti-HIV duoCAR-T cells was investigated using ahumanized intrasplenic NOD-SCID-IL2Rγ−/− mouse model that employs humanPBMC infected with an Env-IMC-LucR HIV-1 virus (hu-spl-PBMC-NSG) toestablish a rapid, strong, and easily quantifiable HIV-1 infection inthe spleens of mice as previously described (Bardhi et al., J Virol2017, 91:20; Thomas et al., Methods Mol Bio 2016, 1354:221-35). Briefly,10 million donor matched PBMCs were activated as described above andspinfected with Du422.1 at 106 IU per 107 PBMCs. HIV-infected PBMCs wereco-injected intrasplenically with either untransduced T-cells (UTD),LTG2303 (bispecific duoCAR), or LTG2330 (trispecific duoCAR) containing30-50% CAR-positive T-cells at an E:T ratio of 0.5:1 (e.g., 5 milliontotal CAR-T cells and 10 million HIV-infected PBMC). PBMC and T cellscontaining CAR effector cells were mixed right before the injections.One week (acute) or one month (chronic) after infection, mice weresacrificed and spleens harvested for further analyses. The totalsplenocytes were separated into three groups to perform the followingassays: 1) luciferase assay to measure infection, 2) flow cytometryanalysis to detect CD4⁺ and CD8⁺ T cells, and 3) DNA extraction forprecise detection of CAR-T cell persistence at the end of the experimentby qPCR. The HIV-1 infection was quantified in a portion of thesplenocytes using the Renilla luciferase assay system (Promega, Madison,Wis.) as previously described Seay et al., Journal of Virology 2015.

In Vivo Persistence of CAR-T Cells Using Real-Time c-Frag qPCR

Briefly, 1 million mouse splenocytes were harvested and genomic DNAextracted by ACGT, Inc. (Germantown, Md.) using the Promega Maxwell® 16LEV Blood DNA Kit. To determine CAR-T persistence, a unique fragment(c-frag) contained within the lentiviral vector backbone of anti-HIVCARs, but not the HIV-1 virus, was used to assess copy numbers per 50 ngof genomic DNA isolated from mouse splenocytes. The following primersand probe were used to detect c-frag: Forward primer:5′GGAGTTGAGACCAGTGTAGT-3′, Reverse primer: 5 ‘-CCACTCCTGACAACTACTCT-3’,Probe: 5′-FAM-CAGTAGGTGAAGGAGTCGTAGTTG-TAMRA-3′. The following primersand probe were used to detect the polypyrimidine tract binding protein 2(PTBP2) gene to control for PCR inhibition: Forward primer:5′-TCTCCATTCCCTATGTTCATGC-3′, Reverse primer:5′-GTTCCCGCAGAATGGTGAGGTG-3′, Probe:5′-JOEATGTTCCTCGGACCAACTTG-BHQ-1-3′. The number of cFrag copies insplenic DNA was calculated using a standard curve consisting of knownquantities of a plasmid DNA containing the c-frag genetic tag followedby normalization to input splenic DNA (0.05 μg) multiplied by the volumeof sample per PCR reaction (12.5 μL).

Results:

A major challenge in the field of HIV immunotherapy is developingtherapies that precisely target and control HIV to a level that preventsviral escape. Similar to combinational anti-retroviral therapies,multiple strategies to target the HIV envelope using an immunotherapyapproach may enable superior control over HIV infection with theadvantage of targeting the latent HIV reservoir, a problem unmet bycurrent drug therapies. To effectively develop such an immunotherapy mayrequire targeting multiple non-overlapping epitopes on the HIV envelopeprotein that are highly conserved with a propensity to reduce viralfitness upon viral escape. To this end, bispecific CARs were engineeredwith domains that target highly conserved regions required for key stepsinvolved in HIV-1 viral entry (mD1.22), co-receptor usage (m36.4), andfusion (C46). The rules that govern bispecific CAR function as itrelates to binder architecture enabled rationale design of a trispecificCAR. Herein describes the in vitro and in vivo efficacy of bispecificand trispecific anti-HIV CARs against genetically diverse and resistantEnv-IMC-LucR HIV-1 viruses encoding env genes found globally. Further tothis, the m36.4 domain and C46 peptide were also evaluated for theirability to protect anti-HIV CARs in vitro and only the most potentanti-HIV CARs evaluated for in vivo efficacy.

As shown in FIG. 13, the donors used in the HIV-1 challenge studies wereenriched for CD4⁺ T cell effectors, the major cellular target for HIV-1.As shown in FIGS. 14A-K, anti-HIV CARs exhibited exceptional potencyagainst all Env-IMC-LucR HIV-1 viruses tested. As expected, themonospecific CAR was the least potent (LTG1944, mD1.22-CAR) followed bya bispecific CAR containing a single CD3 chain (LTG1946). For somedonors and HIV-1 strains tested, the trispecific LTG2323 CAR wasslightly more potent than its bispecific LTG1946 counterpart suggestingperhaps a C46-mediated effect. This enhanced anti-viral effect wasobserved for Env-IMC-LucR viruses encoding the env gene from SF162(Donor I), Du172.17 (Donors H and I), and AE.CNE8 (Donor H). Conversely,LTG2323 was the least potent of the trispecific CARs. As shown in FIG.14 for different donors, the anti-HIV bispecific (LTG2303) andtrispecific CARs (LTG2329, and LTG2330) engineered with two CD3 chains(multi-specific duoCARs) were exceptionally potent and broad againstPBMC infected with several Env-IMC-LucR viruses (See FIG. 15, up to3-log suppression as compared to UTD control). Notably, bispecific andtrispecific duoCAR-T cells also eliminated PBMC infected with severalEnv-IMC-LucR viruses encoding clade C env genes that are resistant toVRC01 and in some instances 3BNC117, two broadly neutralizing antibodiesthat target the CD4 binding site and are currently in clinical trials(Bar et al., N Engl J Med 2016, 375:2037-2050; Liu, Bai, Liu, Zhang, &Wang, Int Immunopharmacol 2017; 52, 44-50). As summarized in FIGS. 15and 16, the bispecific and trispecific duoCAR-T cells were consistentlythe most potent CAR architecture and eliminated up to ˜3 logs or ˜99% ofHIV-1 infection for almost all Env-IMC-LucR viruses tested,respectively. As shown in FIGS. 17A and B, further interrogation ofmulti-specific duoCAR T cells revealed that even at extremely low E:Tratios, for example 1:100, the multi-specific duoCAR-T cellssignificantly eliminated PBMC infected with 3BNC117/VRC01-resistantDu422.1-IMC-LucR virus in contrast to UTD control or conventional CAR-Tcells such as, monospecific 1944 CAR or bispecific 1946 CAR.

In FIG. 18, the mD1.22-CAR T cells were apparently more susceptible toHIV-1 infection during in vitro protection assays than other CARstested. The presence of the m36.4 domain was sufficient to conferprotection of CAR T cells and ablate HIV-1 infection to levelscomparable to uninfected PBMCs. No statistical difference was observedbetween the conventional bispecific 1946 CAR and bispecific 2303 duoCAR(BaL, P=0.06; NL4-3, P=0.09; SF162, P=0.2; AE.CNE55, P=0.9) which bothcontain the m36.4 domain but in different CAR architectures. This resultindicates that the ability of m36.4 to protect CAR-T cells isarchitecture independent. Furthermore, the ability of m36.4 to fullyprotect CART cells and its redundancy to the C46 peptide appears to besufficient for protection. However, it is postulated that in the eventof viral escape (e.g., m36.4 mutational escape), the C46 peptide may berequired for long-term anti-viral efficacy. Hence, it is unclear fromthese data if the C46 peptide is required in addition to the m36.4domain. FIG. 18J shows the compiled luciferase activity data for alldonors and Env-IMC-LucR viruses tested.

To evaluate the in vivo efficacy of CAR-T cells, the bispecific andtrispecific duoCAR-T cells (LTG2303 and LTG2330) were tested using ahumanized intrasplenic HIV infection model (hu-spl-PBMC-NSG) using theDu422.1-IMC-LucR HIV-1 virus as depicted in FIG. 19A. After seven daysof HIV-1 infection, the 2303 and 2330 duoCAR-T cells potently suppressedacute HIV infection in contrast to UTD control and to levels near thatof uninfected PBMCs (FIG. 19B, P=0.003). Although some non-specificactivity was observed for the UTD-treated HIV-infected mice, it was notsufficient to potently eliminate HIV-1 infection as that demonstrated byduoCAR-T cells. In the month-long study, where chronic HIV infection(Du422.1-infected PBMC) persisted in NSG mice, the multi-specificduoCAR-T cells robustly eliminated HIV-infected PBMC in four out of fivemice in contrast to UTD treated, HIV-infected mice (FIG. 19C). Duringacute HIV infection (FIG. 19D), there was no apparent loss of CD4⁺ Tcells for UTD control or CAR-treated HIV-infected mice. However, asshown in FIG. 19E, the CD4⁺ T cells were significantly depleted inchronically-infected mice treated with the UTD control (see UTD+HIVgroup). Conversely, the multi-specific duoCAR-treated, HIV-infected micehad significantly higher percentages of CD4⁺ T cell in their spleens andsimilar to that of uninfected mice (see group HIV-PBMC). In theHIV-infected cohort that did not control HIV infection, as expected,this group (UTD+HIV) also had significantly higher CD8⁺ T cellpercentages (P<0.01, FIG. 19G) concomitant with the loss of CD4⁺ T cells(FIG. 19E) which was not evident in the acute HIV infection study (FIGS.19D and 19F). Lastly, the multi-specific duoCAR-T cells were durable andpersisted during acute (FIG. 19H) and chronic (FIG. 19I) HIV-1infection. Collectively, these data clearly demonstrate the superioradvantage of treating HIV-1 infection with multi-specific anti-HIV CAR Tcells containing the herein described novel domains and novelcompositions. This data coupled with the known long-term persistence andimmunosurveillance properties of CAR-T cells may represent a viableapproach for controlling viral loads and eliminating HIV-infected cellsin HIV positive patients in the absence of anti-retroviral therapy, withthe potential to significantly delay or postpone disease progression.When combined with strategies that target the latent reservoir, anti-HIVduoCAR-T cells could offer a path towards functionally curing HIVinfection.

Each of the applications and patents cited in this text, as well as eachdocument or reference cited in each of the applications and patents(including during the prosecution of each issued patent; “applicationcited documents”), and each of the PCT and foreign applications orpatents corresponding to and/or claiming priority from any of theseapplications and patents, and each of the documents cited or referencedin each of the application cited documents, are hereby expresslyincorporated herein by reference, and may be employed in the practice ofthe invention. More generally, documents or references are cited in thistext, either in a Reference List before the claims, or in the textitself; and, each of these documents or references (“herein citedreferences”), as well as each document or reference cited in each of theherein cited references (including any manufacturer's specifications,instructions, etc.), is hereby expressly incorporated herein byreference.

The foregoing description of some specific embodiments providessufficient information that others can, by applying current knowledge,readily modify or adapt for various applications such specificembodiments without departing from the generic concept, and, therefore,such adaptations and modifications should and are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology employed herein is for the purpose of description and not oflimitation. In the drawings and the description, there have beendisclosed exemplary embodiments and, although specific terms may havebeen employed, they are unless otherwise stated used in a generic anddescriptive sense only and not for purposes of limitation, the scope ofthe claims therefore not being so limited. Moreover, one skilled in theart will appreciate that certain steps of the methods discussed hereinmay be sequenced in alternative order or steps may be combined.Therefore, it is intended that the appended claims not be limited to theparticular embodiment disclosed herein. Those skilled in the art willrecognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the embodiments of the inventiondescribed herein. Such equivalents are encompassed by the followingclaims.

Sequences of the Disclosure

The nucleic and amino acid sequences listed below are shown usingstandard letter abbreviations for nucleotide bases, and three lettercode for amino acids, as defined in 37 C.F.R. 1.822. Only one strand ofeach nucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand. In theaccompanying sequence listing:

SEQ ID NO: 1 is the nucleic acid sequence of the HIV envelope-specific binder mD1.22:AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGGSEQ ID NO: 2 is the amino acid sequence of the HIV envelope-specific binder mD1.22:KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGSEQ ID NO: 3 is the nucleic acid sequence of the HIV envelope-specific binder m36.4:CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASEQ ID NO: 4 is the amino acid sequence of the HIV envelope-specific binder m36.4:QVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLV TVSSSEQ ID NO: 5 is the nucleic acid sequence of the HIV envelope-specific binder C46:TGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCSEQ ID NO: 6 is the amino acid sequence of the HIV envelope-specific binder C46:WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFSEQ ID NO: 7 is the nucleic acid sequence of the CD8 transmembrane domain:ATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCSEQ ID NO: 8 is the amino acid sequence of the CD8 transmembrane domain:IYIWAPLAGTCGVLLLSLVITLYCSEQ ID NO: 9 is the nucleic acid sequence of the CD8 linker/hinge domain:ACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATSEQ ID NO: 10 is the amino acid sequence of the CD8 linker/hinge domain:TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSEQ ID NO: 11 is the nucleic acid sequence of the CD8 linker/hinge/CD8 transmembranedomain: ACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCSEQ ID NO: 12 is the amino acid sequence of the CD8 linker/hinge/CD8 transmembranedomain: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCSEQ ID NO: 13 is the nucleic acid sequence of the TNFRSF19 transmembrane domain:GATACCGCACTGGCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGSEQ ID NO: 14 is the amino acid sequence of the TNFRSF19 transmembrane domain:DTALAAVICSALATVLLALLILCVIYCKRQSEQ ID NO: 15 is the nucleic acid sequence of the CD8 linker linked to the TNFRSF19transmembrane domain (TNFRSF19TM) domain:CCGGCTCCACGACCACCCACTCCAGCCCCAACGATTGCGAGCCAACCTCTCAGTCTTCGGCCCGAGGCTTGCAGGCCAGCCGCAGGAGGAGCAGTGCACACCCGAGGACTGGATTTCGATACCGCACTGGCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGSEQ ID NO: 16 is the amino acid sequence of the CD8 linker linked to the TNFRSF19transmembrane domain (TNFRSF19TM) domain:PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLA LLILCVIYCKRQSEQ ID NO: 17 is the nucleic acid sequence of the 4-1BB signaling domain (41BB):AAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGSEQ ID NO: 18 is the amino acid sequence of the 4-1BB signaling domain (41BB):KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSEQ ID NO: 19 is the nucleic acid sequence of the CD3-zeta signaling domain (CD3zeta):CGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCA AGCACTCCCACCCCGGSEQ ID NO: 20 is the amino acid sequence of the CD3-zeta signaling domain (CD3zeta):RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPRSEQ ID NO: 21 is the nucleic acid sequence of the alternate sequence CD3-zeta signalingdomain (CD3zeta2):CGGGTTAAATTCTCCCGCAGCGCAGACGCACCCGCCTACCAGCAAGGACAGAATCAGCTCTACAACGAACTGAACCTTGGTAGGAGAGAAGAATATGATGTTCTCGACAAGCGCAGAGGGAGAGATCCAGAGATGGGTGGGAAGCCGCAACGCCGGAAAAACCCACAAGAGGGACTGTACAATGAATTGCAGAAAGATAAGATGGCCGAGGCTTACTCAGAAATCGGAATGAAGGGGGAGCGGCGGAGGGGCAAGGGACATGATGGTCTCTACCAAGGGCTTTCAACCGCTACTAAGGACACTTATGACGCACTCCACATGCA GGCGCTGCCTCCGCGASEQ ID NO: 22 is the amino acid sequence of the alternate sequence CD3-zeta signalingdomain (CD3zeta2):RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPRSEQ ID NO: 23 is the nucleic acid sequence of linker domain L1:GGCGGAGGCGGGTCASEQ ID NO: 24 is the amino acid sequence of the linker domain L1: GGGGSSEQ ID NO: 25 is the nucleic acid sequence of linker domain L2:GGCGGAGGCGGGTCAGGTGGCGGTGGTAGTSEQ ID NO: 26 is the amino acid sequence of the linker domain L2:GGGGSGGGGSSEQ ID NO: 27 is the nucleic acid sequence of linker domain L3:GGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCSEQ ID NO: 28 is the amino acid sequence of the linker domain L3:GGGGSGGGGSGGGGSSEQ ID NO: 29 is the nucleic acid sequence of linker domain L4:GGCGGAGGCGGGTCAGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAGGCGGTGGT GGGAGTSEQ ID NO: 30 is the amino acid sequence of the linker domain L4:GGGGSGGGGSGGGGSGGGGSSEQ ID NO: 31 is the nucleic acid sequence of linker domain L5:GGCGGAGGCGGGTCAGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAGGCGGTGGTGGGAGTGGGGGAGGAGGCAGCSEQ ID NO: 32 is the amino acid sequence of the linker domain L5:GGGGSGGGGSGGGGSGGGGSGGGGSSEQ ID NO: 33 is the nucleic acid sequence of the linker and cleavage site domain Furin-2A-Furin (F2AF): CGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGGSEQ ID NO: 34 is the amino acid sequence of the linker and cleavage site domain Furin-2A-Furin (F2AF): RAKRSGSGATNFSLLKQAGDVEENPGPRAKRSEQ ID NO: 35 is the nucleic acid sequence of the leader peptide (LP) derived fromGMCSFR: ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCSEQ ID NO: 36 is the amino acid sequence of the leader peptide (LP) derived fromGMCSFR: MLLLVTSLLLCELPHPAFLLIPDTSEQ ID NO: 37 is the nucleic acid sequence of the leader peptide (LP2) derived from CD59:ATGGGAATTCAGGGGGGTTCCGTGCTCTTTGGCTTGCTCCTGGTCCTGGCAGTGTTTTGTCACTCGGGACACAGCCTGCAGSEQ ID NO: 38 is the amino acid sequence of the leader peptide (LP2) derived from CD59:MGIQGGSVLFGLLLVLAVFCHSGHSLQSEQ ID NO: 39 is the nucleic acid sequence of the anti-HIV CAR LTG1944 (LP-mD1.22-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGGGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 40 is the amino acid sequence of the anti-HIV CAR LTG1944 (LP-mD1.22-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPRSEQ ID NO: 41 is the nucleic acid sequence of the anti-HIV CAR LTG1945 (LP-m36.4-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 42 is the amino acid sequence of the anti-HIV CAR LTG1945 (LP-m36.4-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 43 is the nucleic acid sequence of the anti-HIV CAR LTG2328 (LP-C46-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 44 is the amino acid sequence of the anti-HIV CAR LTG2328 (LP-C46-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPFIPAFLLIPDTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPRSEQ ID NO: 45 is the nucleic acid sequence of the linked anti-HIV binders contained inLTG2325 (mD1.22-L1-m36.4):AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGCGGAGGCGGGTCACAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCT GGTCACCGTCTCCTCASEQ ID NO: 46 is the amino acid sequence of the linked anti-HIV binders contained in CARLTG2325 (mD1.22-L1-m36.4):KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSSEQ ID NO: 47 is the nucleic acid sequence of the anti-HIV CAR LTG2325 (LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGCGGAGGCGGGTCACAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCA CCCCGGTAGSEQ ID NO: 48 is the amino acid sequence of the anti-HIV CAR LTG2325 (LP-mD1.22-L1-m36.4-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALIINIQALPPRSEQ ID NO: 49 is the nucleic acid sequence of the linked anti-HIV binders contained inCAR LTG2313 (mD1.22-L2-m36.4):AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASEQ ID NO: 50 is the amino acid sequence of the linked anti-HIV binders contained in CARLTG2313 (mD1.22-L2-m36.4):KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSSEQ ID NO: 51 is the nucleic acid sequence of the anti-HIV CAR LTG2313 (LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 52 is the amino acid sequence of the anti-HIV CAR LTG2313 (LP-mD1.22-L2-m36.4-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 53 is the nucleic acid sequence of the linked anti-HIV binders contained inLTG1946 (mD1.22-L3-m36.4):AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASEQ ID NO: 54 is the amino acid sequence of the linked anti-HIV binders contained inLTG1946 (mD1.22-L3-m36.4):KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGT LVTVSSSEQ ID NO: 55 is the nucleic acid sequence of the anti-HIV CAR LTG1946 (LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 56 is the amino acid sequence of the anti-HIV CAR LTG1946 (LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 57 is the nucleic acid sequence of the linked anti-HIV binders contained inLTG2326 (mD1.22-L4-m36.4):AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGCGGAGGCGGGTCAGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAGGCGGTGGTGGGAGTCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGT CTCCTCASEQ ID NO: 58 is the amino acid sequence of the linked anti-HIV binders contained inLTG2326 (mD1.22-L4-m36.4):KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEY WGQGTLVTVSSSEQ ID NO: 59 is the nucleic acid sequence of the anti-HIV CAR LTG2326 (LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGCGGAGGCGGGTCAGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAGGCGGTGGTGGGAGTCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 60 is the amino acid sequence of the anti-HIV CAR LTG2326 (LP-mD1.22-L4-m36.4-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 61 is the nucleic acid sequence of the linked anti-HIV binders contained inLTG1947 (mD1.22-L5-m36.4):AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGCGGAGGCGGGTCAGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAGGCGGTGGTGGGAGTGGGGGAGGAGGCAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASEQ ID NO: 62 is the amino acid sequence of the linked anti-HIV binders contained inLTG1947 (mD1.22-L5-m36.4):KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSSEQ ID NO: 63 is the nucleic acid sequence of the anti-HIV CAR LTG1947 (LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGCGGAGGCGGGTCAGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAGGCGGTGGTGGGAGTGGGGGAGGAGGCAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGA TACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 64 is the amino acid sequence of the anti-HIV CAR LTG1947 (LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGFIDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 65 is the nucleic acid sequence of the linked anti-HIV binders contained inLTG1948 (m36.4-L3-mD1.22):CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCSEQ ID NO: 66 is the amino acid sequence of the linked anti-HIV binders contained inLTG1948 (m36.4-L3-mD1.22):QVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQ LVVVGSEQ ID NO: 67 is the nucleic acid sequence of the anti-HIV CAR LTG1948 (LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 68 is the amino acid sequence of the anti-HIV CAR LTG1948 (LP-m36.4-L3-mD1.22-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 69 are the nucleic acid sequences of the anti-HIV binders contained inLTG2303 and LTG2322, expressed together in a single cell (mD1.22 AND m36.4):AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGG-CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASEQ ID NO: 70 are the amino acid sequences of the anti-HIV binders contained inLTG2303 and LTG2322, expressed together in a single cell (mD1.22 AND m36.4):KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVG-QVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLV TVSSSEQ ID NO: 71 is the nucleic acid sequence of the anti-HIV CAR LTG2303 (LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGGGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGCGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGGATGGGAATTCAGGGGGGTTCCGTGCTCTTTGGCTTGCTCCTGGTCCTGGCAGTGTTTTGTCACTCGGGACACAGCCTGCAGCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCACCGGCTCCACGACCACCCACTCCAGCCCCAACGATTGCGAGCCAACCTCTCAGTCTTCGGCCCGAGGCTTGCAGGCCAGCCGCAGGAGGAGCAGTGCACACCCGAGGACTGGATTTCGATACCGCACTGGCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGCGGGTTAAATTCTCCCGCAGCGCAGACGCACCCGCCTACCAGCAAGGACAGAATCAGCTCTACAACGAACTGAACCTTGGTAGGAGAGAAGAATATGATGTTCTCGACAAGCGCAGAGGGAGAGATCCAGAGATGGGTGGGAAGCCGCAACGCCGGAAAAACCCACAAGAGGGACTGTACAATGAATTGCAGAAAGATAAGATGGCCGAGGCTTACTCAGAAATCGGAATGAAGGGGGAGCGGCGGAGGGGCAAGGGACATGATGGTCTCTACCAAGGGCTTTCAACCGCTACTAAGGACACTTATGACGCACTCCACATGCAGGCGCTGCC TCCGCGATAASEQ ID NO: 72 is the amino acid sequence of the anti-HIV CAR LTG2303 (LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPRAKRMGIQGGSVLFGLLLVLAVFCHSGHSLQQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLALLILCVIYCKRQRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 73 is the nucleic acid sequence of the anti-HIV CAR LTG2322 (LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF 19TM):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGGGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGCGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGGATGGGAATTCAGGGGGGTTCCGTGCTCTTTGGCTTGCTCCTGGTCCTGGCAGTGTTTTGTCACTCGGGACACAGCCTGCAGCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCACCGGCTCCACGACCACCCACTCCAGCCCCAACGATTGCGAGCCAACCTCTCAGTCTTCGGCCCGAGGCTTGCAGGCCAGCCGCAGGAGGAGCAGTGCACACCCGAGGACTGGATTTCGATACCGCACTGGCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGTAGSEQ ID NO: 74 is the amino acid sequence of the anti-HIV CAR LTG2322 (LP-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPRAKRMGIQGGSVLFGLLLVLAVFCHSGHSLQQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLALLILCVIYCKRQSEQ ID NO: 75 are the nucleic acid sequences of the linked anti-HIV binders in LTG2314(mD1.22-L3-C46): AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCSEQ ID NO: 76 are the amino acid sequences of the linked anti-HIV binders in LTG2314(mD1.22-L3-C46):KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFSEQ ID NO: 77 is the nucleic acid sequence of the anti-HIV CAR LTG2314 (LP-mD1.22-L3-C46-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACT CCCACCCCGGTAGSEQ ID NO: 78 is the amino acid sequence of the anti-HIV CAR LTG2314 (LP-mD1.22-L3-C46-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCScRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPRSEQ ID NO: 79 are the nucleic acid sequences of the linked anti-HIV binders in LTG2315(mD1.22-L5-C46): AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACccAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGCGGAGGCGGGTCAGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAGGCGGTGGTGGGAGTGGGGGAGGAGGCAGCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCSEQ ID NO: 80 are the amino acid sequences of the linked anti-HIV binders in LTG2315(mD1.22-L5-C46):KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSGGGGSGGGGSWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWN WFSEQ ID NO: 81 is the nucleic acid sequence of the anti-HIV CAR LTG2315 (LP-mD1.22-L5-C46-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGCGGAGGCGGGTCAGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAGGCGGTGGTGGGAGTGGGGGAGGAGGCAGCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 82 is the amino acid sequence of the anti-HIV CAR LTG2315 (LP-mD1.22-L5-C46-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSGGGGSGGGGSWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 83 are the nucleic acid sequence of the linked anti-HIV binders in LTG2316expressed in a single cell (C46-L3-mD1.22):TGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGCGGAGGGGGGAGTGGAGGTGGCGGTTCAGGAGGTGGGGGAAGCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCSEQ ID NO: 84 are the amino acid sequences of the linked anti-HIV binders in LTG2316(C46-L3-mD1.22): WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGSEQ ID NO: 85 is the nucleic acid sequence of the anti-HIV CAR LTG2316 (LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGCGGAGGGGGGAGTGGAGGTGGCGGTTCAGGAGGTGGGGGAAGCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 86 is the amino acid sequence of the anti-HIV CAR LTG2316 (LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALFIMQALPPRSEQ ID NO: 87 are the nucleic acid sequence of the linked anti-HIV binders in LTG2317expressed in a single cell (C46-L5-mD1.22):TGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGCGGAGGGGGGAGTGGAGGTGGCGGTTCAGGAGGTGGGGGAAGCGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCSEQ ID NO: 88 are the amino acid sequences of the linked anti-HIV binders in LTG2317(C46-L5-mD1.22): WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLV VVGSEQ ID NO: 89 is the nucleic acid sequence of the anti-HIV CAR LTG2317 (LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGCGGAGGGGGGAGTGGAGGTGGCGGTTCAGGAGGTGGGGGAAGCGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGTAGSEQ ID NO: 90 is the amino acid sequence of the anti-HIV CAR LTG2317 (LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 91 are the nucleic acid sequences of the linked anti-HIV binders in LTG2318(mD1.22-L3-m36.4-L3-C46):AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGTGGAGGGGGCTCTGGCGGTGGAGGGTCCGGGGGAGGTGGCTCGTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCC CTGTGGAACTGGTTCSEQ ID NO: 92 are the amino acid sequences of the linked anti-HIV binders in LTG2318(mD1.22-L3-m36.4-L3-C46):KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSGGGGSGGGGSGGGGSWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEL DKWASLWNWFSEQ ID NO: 93 is the nucleic acid sequence of the anti-HIV CAR LTG2318 (LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGTGGAGGGGGCTCTGGCGGTGGAGGGTCCGGGGGAGGTGGCTCGTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACC CCGGTAGSEQ ID NO: 94 is the amino acid sequence of the anti-HIV CAR LTG2318 (LP-mD1.22-L3-m36.4-L3-C46-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSGGGGSGGGGSGGGGSWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 95 is the nucleic acid sequence of the linked anti-HIV binders in LTG2319(mD1.22-L3-C46-L3-m36.4):AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGTGGAGGGGGCTCTGGCGGTGGAGGGTCCGGGGGAGGTGGCTCGCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTG GTCACCGTCTCCTCASEQ ID NO: 96 is the amino acid sequence of the linked anti-HIV binders in LTG2319(mD1.22-L3-C46-L3-m36.4)KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGE YWGQGTLVTVSSSEQ ID NO: 97 is the nucleic acid sequence of the anti-HIV CAR LTG2319 (LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGTGGAGGGGGCTCTGGCGGTGGAGGGTCCGGGGGAGGTGGCTCGCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCC CGGTAGSEQ ID NO: 98 is the amino acid sequence of the anti-HIV CAR LTG2319 (LP-mD1.22-L3-C46-L3-m36.4-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 99 is the nucleic acid sequence of the linked anti-HIV binders in LTG2320(C46-L3-mD1.22-L3-m36.4):TGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGTGGAGGGGGCTCTGGCGGTGGAGGGTCCGGGGGAGGTGGCTCGAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGG TCACCGTCTCCTCASEQ ID NO: 100 is the amino acid sequence of the linked anti-HIV binders in LTG2320(C46-L3-mD1.22-L3-m36.4):WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGE YWGQGTLVTVSSSEQ ID NO: 101 is the nucleic acid sequence of the anti-HIV CAR LTG2320 (LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGTGGAGGGGGCTCTGGCGGTGGAGGGTCCGGGGGAGGTGGCTCGAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCAC CCCGGTAGSEQ ID NO: 102 is the amino acid sequence of the anti-HIV CAR LTG2320 (LP-C46-L3-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta):MLLLVTSLLLCELPHPAFLLIPDTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 103 is the nucleic acid sequences of the anti-HIV binders contained inLTG2323 (mD1.22-L3-m36.4 and C46) expressed in the same cell:AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGGGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA-TGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCSEQ ID NO: 104 is the amino acid sequences of the anti-HIV binders contained inLTG2323 (mD1.22-L3-m36.4 and C46) expressed in the same cell:KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGT LVTVSS-WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFSEQ ID NO: 105 is the nucleic acid sequence of the anti-HIV CAR LTG2323 (LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-LP2-C46-TNFRSF19TM):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGGGGAGGTGGCGGTTCAGGCGGAGGGGGGAGTGGAGGTGGGGGAAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGCGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGGATGGGAATTCAGGGGGGTTCCGTGCTCTTTGGCTTGCTCCTGGTCCTGGCAGTGTTTTGTCACTCGGGACACAGCCTGCAGTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCCCGGCTCCACGACCACCCACTCCAGCCCCAACGATTGCGAGCCAACCTCTCAGTCTTCGGCCCGAGGCTTGCAGGCCAGCCGCAGGAGGAGCAGTGCACACCCGAGGACTGGATTTCGATACCGCACTGGCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGTAGSEQ ID NO: 106 is the amino acid sequence of the anti-HIV CAR LTG2323 (LP-mD1.22-L3-m36.4-CD8TM-41BB-CD3zeta-F2AF-LP2-C46-TNFRSF19TM):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPRAKRMGIQGGSVLFGLLLVLAVFCHSGHSLQWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLALLILCVIYCKRQSEQ ID NO: 107 is the nucleic acid sequence of the anti-HIV CAR LTG2329 (LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGCGGAGGGGGGAGTGGAGGTGGCGGTTCAGGAGGTGGGGGAAGCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGCGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGGATGGGAATTCAGGGGGGTTCCGTGCTCTTTGGCTTGCTCCTGGTCCTGGCAGTGTTTTGTCACTCGGGACACAGCCTGCAGCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCACCGGCTCCACGACCACCCACTCCAGCCCCAACGATTGCGAGCCAACCTCTCAGTCTTCGGCCCGAGGCTTGCAGGCCAGCCGCAGGAGGAGCAGTGCACACCCGAGGACTGGATTTCGATACCGCACTGGCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGCGGGTTAAATTCTCCCGCAGCGCAGACGCACCCGCCTACCAGCAAGGACAGAATCAGCTCTACAACGAACTGAACCTTGGTAGGAGAGAAGAATATGATGTTCTCGACAAGCGCAGAGGGAGAGATCCAGAGATGGGTGGGAAGCCGCAACGCCGGAAAAACCCACAAGAGGGACTGTACAATGAATTGCAGAAAGATAAGATGGCCGAGGCTTACTCAGAAATCGGAATGAAGGGGGAGCGGCGGAGGGGCAAGGGACATGATGGTCTCTACCAAGGGCTTTCAACCGCTACTAAGGACACTTATGACGCACTCCACATGCAGGCGCTGCCTCCGCGATAASEQ ID NO: 108 is the amino acid sequence of the anti-HIV CAR LTG2329 (LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2):MLLLVTSLLLCELPHPAFLLIPDTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALFIMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPRAKRMGIQGGSVLFGLLLVLAVFCHSGHSLQQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLALLILCVIYCKRQRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPRSEQ ID NO: 109 is the nucleic acid sequence of the anti-HIV CAR LTG2330 (LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4TNFRSF19TM-CD3zeta2):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGCGGAGGGGGGAGTGGAGGTGGCGGTTCAGGAGGTGGGGGAAGCGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGCGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGGATGGGAATTCAGGGGGGTTCCGTGCTCTTTGGCTTGCTCCTGGTCCTGGCAGTGTTTTGTCACTCGGGACACAGCCTGCAGCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCACCGGCTCCACGACCACCCACTCCAGCCCCAACGATTGCGAGCCAACCTCTCAGTCTTCGGCCCGAGGCTTGCAGGCCAGCCGCAGGAGGAGCAGTGCACACCCGAGGACTGGATTTCGATACCGCACTGGCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGCGGGTTAAATTCTCCCGCAGCGCAGACGCACCCGCCTACCAGCAAGGACAGAATCAGCTCTACAACGAACTGAACCTTGGTAGGAGAGAAGAATATGATGTTCTCGACAAGCGCAGAGGGAGAGATCCAGAGATGGGTGGGAAGCCGCAACGCCGGAAAAACCCACAAGAGGGACTGTACAATGAATTGCAGAAAGATAAGATGGCCGAGGCTTACTCAGAAATCGGAATGAAGGGGGAGCGGCGGAGGGGCAAGGGACATGATGGTCTCTACCAAGGGCTTTCAACCGCTACTAAGGACACTTATGACGCACTCCACATGCAGGCGCTGCC TCCGCGATAASEQ ID NO: 110 is the amino acid sequence of the anti-HIV CAR LTG2330 (LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM-CD3zeta2):MLLLVTSLLLCELPHPAFLLIPDTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPRAKRMGIQGGSVLFGLLLVLAVFCHSGHSLQQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLALLILCVIYCKRQRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 111 is the nucleic acid sequences of the anti-HIV binders contained inLTG2329 and LTG2331 (C46-L3-mD1.22 and m36.4) expressed in the same cell:TGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGCGGAGGGGGGAGTGGAGGTGGCGGTTCAGGAGGTGGGGGAAGCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGC-CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGT AACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASEQ ID NO: 112 is the amino acid sequences of the anti-HIV binders contained inLTG2329 and LTG2331 (C46-L3-mD1.22 and m36.4) expressed in the same cell:WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVG-QVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLV TVSSSEQ ID NO: 113 is the nucleic acid sequence of the anti-HIV CAR LTG2331 (LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGCGGAGGGGGGAGTGGAGGTGGCGGTTCAGGAGGTGGGGGAAGCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGCGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGGATGGGAATTCAGGGGGGTTCCGTGCTCTTTGGCTTGCTCCTGGTCCTGGCAGTGTTTTGTCACTCGGGACACAGCCTGCAGCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCACCGGCTCCACGACCACCCACTCCAGCCCCAACGATTGCGAGCCAACCTCTCAGTCTTCGGCCCGAGGCTTGCAGGCCAGCCGCAGGAGGAGCAGTGCACACCCGAGGACTGGATTTCGATACCGCACTGGCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGTAGSEQ ID NO: 114 is the amino acid sequence of the anti-HIV CAR LTG2331 (LP-C46-L3-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM):MLLLVTSLLLCELPHPAFLLIPDTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALFIMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPRAKRMGIQGGSVLFGLLLVLAVFCHSGHSLQQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLALLILCVIYCKRQSEQ ID NO: 115 is the nucleic acid sequences of the anti-HIV binders contained inLTG2330 and LTG2332 (C46-L5-mD1.22 and m36.4) expressed in the same cell:TGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGCGGAGGGGGGAGTGGAGGTGGCGGTTCAGGAGGTGGGGGAAGCGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGC-CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGT AACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCASEQ ID NO: 116 is the amino acid sequences of the anti-HIV binders contained inLTG2330 and LTG2332 (C46-L5-mD1.22 and m36.4) expressed in the same cell:WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLV VVG-QVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLV TVSSSEQ ID NO: 117 is the nucleic acid sequence of the anti-HIV CAR LTG2332 (LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCGGCGGAGGGGGGAGTGGAGGTGGCGGTTCAGGAGGTGGGGGAAGCGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGCGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGGATGGGAATTCAGGGGGGTTCCGTGCTCTTTGGCTTGCTCCTGGTCCTGGCAGTGTTTTGTCACTCGGGACACAGCCTGCAGCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCACCGGCTCCACGACCACCCACTCCAGCCCCAACGATTGCGAGCCAACCTCTCAGTCTTCGGCCCGAGGCTTGCAGGCCAGCCGCAGGAGGAGCAGTGCACACCCGAGGACTGGATTTCGATACCGCACTGGCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGTAGSEQ ID NO: 118 is the amino acid sequence of the anti-HIV CAR LTG2332 (LP-C46-L5-mD1.22-CD8TM-41BB-CD3zeta-F2AF-LP2-m36.4-TNFRSF19TM):MLLLVTSLLLCELPHPAFLLIPDTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFGGGGSGGGGSGGGGSGGGGSGGGGSKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPRAKRMGIQGGSVLFGLLLVLAVFCHSGHSLQQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLALLILCVIYCKRQSEQ ID NO: 119 is the nucleic acid sequence of the anti-HIV binders contained inLTG2334 (mD1.22-L5-m36.4 and C46) expressed in the same cell:AAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGCGGAGGCGGGTCAGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAGGCGGTGGTGGGAGTGGGGGAGGAGGCAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCA-TGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCSEQ ID NO: 120 is the amino acid sequence of the anti-HIV binders contained in LTG2334(mD1.22-L5-m36.4 and C46) expressed in the same cell:KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSS- WMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFSEQ ID NO: 121 is the nucleic acid sequence of the anti-HIV CAR LTG2334 (LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-LP2-C46-TNFRSF19TM):ATGCTTCTCCTGGTCACCTCCCTGCTCCTCTGCGAACTGCCTCACCCTGCCTTCCTTCTGATTCCTGACACCAAGAAAGTCGTGTACGGAAAGAAGGGAGACACTGTGGAGCTGACCTGTACCGCAAGCCAGAAGAAGAACATCCAGTTCCACTGGAAGAACTCCAACCAAATCAAGATCCTGGGGAACCAGGGTTCCTTCCTGACTAAGGGACCCTCAAAGCTGAACGACCGCGTGGATAGCAGACGCTCCCTGTGGGACCAGGGAAACTTCCCGCTTATCATTAAGAACCTCAAACCTGAGGACTCGGATACCTACATCTGCGAAGTGGAGGACCAGAAGGAGGAGGTGCAGCTGGTGGTGGTGGGCGGCGGAGGCGGGTCAGGTGGCGGTGGTAGTGGCGGTGGCGGTTCAGGCGGTGGTGGGAGTGGGGGAGGAGGCAGCCAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGCTTTCGATTTCTCTGATTATGAAATGAGCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATGATAGTGGAAACACCATTTACAATCCGTCCCTCAAGAGTCGAGTCACCATCTCCAGAGACAATTCCAAGAACACACTGTATCTGCAAATGAACACCCTGAGAGCCGAGGACACAGCCATATATTACTGTGCGATATATGGTGGTAACTCCGGGGGAGAGTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCGGCCGCAACTACCACCCCTGCCCCTCGGCCGCCGACTCCGGCCCCAACCATCGCAAGCCAACCCCTCTCCTTGCGCCCCGAAGCTTGCCGCCCGGCCGCGGGTGGAGCCGTGCATACCCGGGGGCTGGACTTTGCCTGCGATATCTACATTTGGGCCCCGCTGGCCGGCACTTGCGGCGTGCTCCTGCTGTCGCTGGTCATCACCCTTTACTGCAAGAGGGGCCGGAAGAAGCTGCTTTACATCTTCAAGCAGCCGTTCATGCGGCCCGTGCAGACGACTCAGGAAGAGGACGGATGCTCGTGCAGATTCCCTGAGGAGGAAGAGGGGGGATGCGAACTGCGCGTCAAGTTCTCACGGTCCGCCGACGCCCCCGCATATCAACAGGGCCAGAATCAGCTCTACAACGAGCTGAACCTGGGAAGGAGAGAGGAGTACGACGTGCTGGACAAGCGACGCGGACGCGACCCGGAGATGGGGGGGAAACCACGGCGGAAAAACCCTCAGGAAGGACTGTACAACGAACTCCAGAAAGACAAGATGGCGGAAGCCTACTCAGAAATCGGGATGAAGGGAGAGCGGAGGAGGGGAAAGGGTCACGACGGGCTGTACCAGGGACTGAGCACCGCCACTAAGGATACCTACGATGCCTTGCATATGCAAGCACTCCCACCCCGGCGCGCGAAACGCAGCGGCAGCGGCGCGACCAACTTTAGCCTGCTGAAACAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGCGAGCAAAGAGGATGGGAATTCAGGGGGGTTCCGTGCTCTTTGGCTTGCTCCTGGTCCTGGCAGTGTTTTGTCACTCGGGACACAGCCTGCAGTGGATGGAATGGGATCGCGAAATCAACAACTACACCTCCCTGATTCACTCCCTGATTGAGGAATCCCAGAATCAACAGGAGAAGAACGAACAAGAGCTTCTGGAGCTGGACAAATGGGCCTCCCTGTGGAACTGGTTCCCGGCTCCACGACCACCCACTCCAGCCCCAACGATTGCGAGCCAACCTCTCAGTCTTCGGCCCGAGGCTTGCAGGCCAGCCGCAGGAGGAGCAGTGCACACCCGAGGACTGGATTTCGATACCGCACTGGCGGCCGTGATCTGTAGCGCCCTGGCCACCGTGCTGCTGGCGCTGCTCATCCTTTGCGTGATCTACTGCAAGCGGCAGTAGSEQ ID NO: 122 is the amino acid sequence of the anti-HIV CAR LTG2334 (LP-mD1.22-L5-m36.4-CD8TM-41BB-CD3zeta-F2AF-LP2-C46-TNFRSF19TM):MLLLVTSLLLCELPHPAFLLIPDTKKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIKNLKPEDSDTYICEVEDQKEEVQLVVVGGGGGSGGGGSGGGGSGGGGSGGGGSQVQLVQSGGGLVQPGGSLRLSCAASAFDFSDYEMSWVREAPGKGLEWIGEINDSGNTIYNPSLKSRVTISRDNSKNTLYLQMNTLRAEDTAIYYCAIYGGNSGGEYWGQGTLVTVSSAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAKRSGSGATNFSLLKQAGDVEENPGPRAKRMGIQGGSVLFGLLLVLAVFCHSGHSLQWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFDTALAAVICSALATVLLALLILCVIYCKRQ

1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising at least one extracellular antigen binding domain comprising an anti-HIV envelope antigen binding domain encoded by a nucleotide sequence comprising SEQ ID NO. 1, 3, 5, 45, 49, 53, 57, 61, 65, 69, 75, 79, 83, 87, 91, 95, 99, 103, 111, 115, or 119, at least one transmembrane domain, and at least one intracellular signaling domain. 2.-21. (canceled)
 22. A vector comprising a nucleic acid molecule of claim
 1. 23. The vector of claim 22, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, adenoviral vector, and a retrovirus vector, or a combination thereof. 24.-25. (canceled)
 26. A cell comprising the vector of claim
 22. 27.-30. (canceled)
 31. A method of making a cell comprising transducing a T cell with a vector of claim
 22. 32.-34. (canceled)
 35. A pharmaceutical composition comprising an anti-HIV effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain comprising an anti-HIV antigen binding domain comprising the amino acid sequence of SEQ ID NO. 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, or 120, at least one linker domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are T cells of a human having a HIV/AIDS. 36.-38. (canceled)
 39. A method of treating HIV-related cancers or HIV/AIDS in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells, wherein the T cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain comprising an anti-HIV antigen binding domain comprising the amino acid sequence of SEQ ID NO. 2, 4, 6, 46, 50, 54, 58, 62, 66, 70, 76, 80, 84, 88, 92, 96, 100, 104, 112, 116, or 120, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, wherein the T cells are T cells of the subject having HIV/AIDS.
 40. The method of claim 39, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta or zeta chain of the T-cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or any combination thereof. 41.-42. (canceled)
 43. The method of claim 39, wherein the subject has cancer, and the cancer is a HIV-related cancer.
 44. The method of claim 39, wherein the anti-HIV antigen binding protein binds to HIV-1 envelope protein.
 45. The method of claim 39, wherein the anti-HIV antigen binding domain, the at least one intracellular signaling domain, or both are connected to the at least one transmembrane domain by a linker or spacer domain.
 46. The method of claim 39, wherein the linker or spacer domain is derived from the extracellular domain of CD8, and is linked to a transmembrane domain.
 47. The method of claim 39, wherein the at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.
 48. The method of claim 39, wherein the nucleic acid sequence encoding the extracellular anti-HIV antigen binding domain comprises a nucleic sequence comprising SEQ ID NO. 1, 3, 5, 45, 49, 53, 57, 61, 65, 69, 75, 79, 83, 87, 91, 95, 99, 103, 111, 115, and 119, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.
 49. The method of claim 39, wherein the at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof.
 50. The method of claim 48, wherein the at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of: OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.
 51. The method of claim 48, wherein the nucleic acid sequence encoding the extracellular anti-HIV antigen binding domain comprises a nucleic sequence comprising SEQ ID NO. 69, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.
 52. The method of claim 48, wherein the nucleic acid sequence encoding the extracellular anti-HIV antigen binding domain comprises a nucleic sequence comprising SEQ ID NO. 115, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.
 53. The method of claim 39, wherein the at least one extracellular antigen binding domain comprising an anti-HIV antigen binding domain comprising the amino acid sequence of SEQ ID NO.
 70. 54. The method of claim 39, wherein the at least one extracellular antigen binding domain comprising an anti-HIV antigen binding domain comprising the amino acid sequence of SEQ ID NO.
 116. 